FGF-CX polynucleotide sequences and methods of producing same

ABSTRACT

The present invention provides FGF-CX, a novel isolated polypeptide, as well as a polynucleotide encoding FGF-CX and antibodies that immunospecifically bind to FGF-CX or any derivative, variant, mutant, or fragment of the FGF-CX polypeptide, polynucleotide or antibody. The invention additionally provides methods in which the FGF-GX polypeptide, polynucleotide and antibody are used in detection and treatment of a broad range of pathological states, as well as to other uses.

RELATED APPLICATION

This application claims priority to U.S. Provisional patent applicationSer. No. 60/145,899, entitled “Novel FGF-Like Protein,” filed Jul. 27,1999, and is a continuation application of U.S. patent application Ser.No. 09/494,585, filed Jan. 31, 2000 now abandoned. The contents of thesepriority applications are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The invention generally relates to nucleic acids and polypeptides. Theinvention relates more particularly to nucleic acids encodingpolypeptides related to a novel member of the fibroblast growth factorfamily.

BACKGROUND OF THE INVENTION

The FGF family of proteins, whose prototypic members include acidic FGF(FGF-1) and basic FGF (FGF-2), bind to four related receptor tyrosinekinases. These FGF receptors are expressed on most types of cells intissue culture. Dimerization of FGF receptor monomers upon ligandbinding has been reported to be a requisite for activation of the kinasedomains, leading to receptor trans phosphorylation. FGF receptor-1(FGFR-1), which shows the broadest expression pattern of the four FGFreceptors, contains at least seven tyrosine phosphorylation sites. Anumber of signal transduction molecules are affected by binding withdifferent affinities to these phosphorylation sites.

Expression of FGFs and their receptors in brains of perinatal and adultmice has been examined. Messenger RNA all FGF genes, with the exceptionof FGF4, is detected in these tissues. FGF-3, FGF-6, FGF-7 and FGF-8genes demonstrate higher expression in the late embryonic stages than inpostnatal stages, suggesting that these members are involved in the latestages of brain development. In contrast, expression of FGF-1 and FGF-5increased after birth. In particular, FGF-6 expression in perinatal micehas been reported to be restricted to the central nervous system andskeletal muscles, with intense signals in the developing cerebrum inembryos but in cerebellum in 5-day-old neonates. FGF-receptor (FGFR)4, acognate receptor for FGF-6, demonstrate similar spatiotemporalexpression, suggesting that FGF-6 and FGFR4 plays significant roles inthe maturation of nervous system as a ligand-receptor system. Accordingto Ozawa et al., these results strongly suggest that the various FGFsand their receptors are involved in the regulation of a variety ofdevelopmental processes of brain, such as proliferation and migration ofneuronal progenitor cells, neuronal and glial differentiation, neuriteextensions, and synapse formation.

Other members of the FGF polypeptide family include the FGF receptortyrosine kinase (FGFRTK) family and the FGF receptor heparan sulfateproteoglycan (FGFRHS) family. These members interact to regulate activeand specific FGFR signal transduction complexes. These regulatoryactivities are diversified throughout a broad range of organs andtissues, and in both normal and tumor tissues, in mammals. Regulatedalternative messenger RNA (mRNA) splicing and combination of variantsubdomains give rise to diversity of FGFRTK monomers. Divalent cationscooperate with the FGFRHS to conformationally restrict FGFRTKtrans-phosphorylation, which causes depression of kinase activity andfacilitates appropriate activation of the FGFR complex by FGF. Forexample, it is known that different point mutations in the FGFRTKcommonly cause craniofacial and skeletal abnormalities of gradedseverity by graded increases in FGF-independent activity of total FGFRcomplexes. Other processes in which FGF family exerts important effectsare liver growth and function and prostate tumor progression.

Glia-activating factor (GAF), another FGF family member, is aheparin-binding growth factor that was purified from the culturesupernatant of a human glioma cell line. See, Miyamoto et al., 1993, MolCell Biol 13(7): 4251-4259. GAF shows a spectrum of activity slightlydifferent from those of other known growth factors, and is designated asFGF-9. The human FGF-9 cDNA encodes a polypeptide of 208 amino acids.Sequence similarity to other members of the FGF family was estimated tobe around 30%. Two cysteine residues and other consensus sequences foundin other family members were also well conserved in the FGF-9 sequence.FGF-9 was found to have no typical signal sequence in its N terminuslike those in acidic FGF and basic FGF. Acidic FGF and basic FGF areknown not to be secreted from cells in a conventional manner. However,FGF-9 was found to be secreted efficiently from cDNA-transfected COScells despite its lack of a typical signal sequence. It could bedetected exclusively in the culture medium of cells. The secretedprotein lacked no amino acid residues at the N terminus with respect tothose predicted by the cDNA sequence, except the initiation methionine.The rat FGF-9 cDNA was also cloned, and the structural analysisindicated that the FGF-9 gene is highly conserved.

SUMMARY OF THE INVENTION

The present invention is based, in part, upon the discovery of a nucleicacid encoding a novel polypeptide having homology to Fibroblast GrowthFactor (FGF) protein. Fibroblast Grown Factor-CX (FGF-CX) polynucleotidesequences and the FGF-CX polypeptides encoded by these nucleic acidsequences, and fragments, homologs, analogs, and derivatives thereof,are claimed in the invention.

In one aspect, the invention provides an isolated FGF-CX nucleic acid(SEQ ID NO:1, as shown in FIG. 1), that encodes a FGF-CX polypeptide, ora fragment, homolog, analog or derivative thereof. The nucleic acid caninclude, e.g., nucleic acid sequence encoding a polypeptide at least 85%identical to a polypeptide comprising the amino acid sequence of FIG. 1(SEQ ID NO:2). The nucleic acid can be, e.g., a genomic DNA fragment, orit can be a cDNA molecule.

Also included in the invention is a vector containing one or more of thenucleic acids described herein, and a cell containing the vectors ornucleic acids described herein.

The present invention is also directed to host cells transformed with arecombinant expression vector comprising any of the nucleic acidmolecules described above.

In one aspect, the invention includes a pharmaceutical composition thatincludes a FGF-CX nucleic acid and a pharmaceutically acceptable carrieror diluent. In a further aspect, the invention includes a substantiallypurified FGF-CX polypeptide, e.g., any of the FGF-CX polypeptidesencoded by a FGF-CX nucleic acid, and fragments, homologs, analogs, andderivatives thereof. The invention also includes a pharmaceuticalcomposition that includes a FGF-CX polypeptide and a pharmaceuticallyacceptable carrier or diluent.

In a further aspect, the invention provides an antibody that bindsspecifically to a FGF-CX polypeptide. The antibody can be, e.g., amonoclonal or polyclonal antibody, and fragments, homologs, analogs, andderivatives thereof. The invention also includes a pharmaceuticalcomposition including FGF-CX antibody and a pharmaceutically acceptablecarrier or diluent. The present invention is also directed to isolatedantibodies that bind to an epitope on a polypeptide encoded by any ofthe nucleic acid molecules described above.

The present invention is further directed to kits comprising antibodiesthat bind to a polypeptide encoded by any of the nucleic acid moleculesdescribed above and a negative control antibody.

The invention further provides a method for producing a FGF-CXpolypeptide. The method includes providing a cell containing a FGF-CXnucleic acid, e.g., a vector that includes a FGF-CX nucleic acid, andculturing the cell under conditions sufficient to express the FGF-CXpolypeptide encoded by the nucleic acid. The expressed FGF-CXpolypeptide is then recovered from the cell. Preferably, the cellproduces little or no endogenous FGF-CX polypeptide. The cell can be,e.g., a prokaryotic cell or eukaryotic cell.

The present invention provides a method of inducing an immune responsein a mammal against a polypeptide encoded by any of the nucleic acidmolecules disclosed above by administering to the mammal an amount ofthe polypeptide sufficient to induce the immune response.

The present invention is also directed to methods of identifying acompound that binds to FGF-CX polypeptide by contacting the FGF-CXpolypeptide with a compound and determining whether the compound bindsto the FGF-CX polypeptide.

The invention further provides methods of identifying a compound thatmodulates the activity of a FGF-CX polypeptide by contacting FGF-CXpolypeptide with a compound and determining whether the FGF-CXpolypeptide activity is modified.

The present invention is also directed to compounds that modulate FGF-CXpolypeptide activity identified by contacting a FGF-CX polypeptide withthe compound and determining whether the compound modifies activity ofthe FGF-CX polypeptide, binds to the FGF-CX polypeptide, or binds to anucleic acid molecule encoding a FGF-CX polypeptide.

In another aspect, the invention provides a method of diagnosing atissue proliferation-associated disorder, such as tumors, restenosis,psoriasis, diabetic and post-surgery complications, and rheumatoidarthritis, in a subject. The method includes providing a protein samplefrom the subject and measuring the amount of FGF-CX polypeptide in thesubject sample. The amount of FGF-CX in the subject sample is thencompared to the amount of FGF-CX polypeptide in a control proteinsample. An alteration in the amount of FGF-CX polypeptide in the subjectprotein sample relative to the amount of FGF-CX polypeptide in thecontrol protein sample indicates the subject has a tissueproliferation-associated condition. A control sample is preferably takenfrom a matched individual, i.e., an individual of similar age, sex, orother general condition but who is not suspected of having a tissueproliferation-associated condition. Alternatively, the control samplemay be taken from the subject at a time when the subject is notsuspected of having a tissue proliferation-associated disorder. In someembodiments, the FGF-CX polypeptide is detected using a FGF-CX antibody.

The invention is also directed to methods of inducing an immune responsein a mammal against a polypeptide encoded by any of the nucleic acidmolecules described above. The method includes administering to themammal an amount of the polypeptide sufficient to induce the immuneresponse.

In a further aspect, the invention includes a method of diagnosing atissue proliferation-associated disorder, such as tumors, restenosis,psoriasis, diabetic and post-surgery complications, and rheumatoidarthritis, in a subject. The method includes providing a nucleic acidsample, e.g., RNA or DNA, or both, from the subject and measuring theamount of the FGF-CX nucleic acid in the subject nucleic acid sample.The amount of FGF-CX nucleic acid sample in the subject nucleic acid isthen compared to the amount of FGF-CX nucleic acid in a control sample.An alteration in the amount of FGF-CX nucleic acid in the samplerelative to the amount of FGF-CX in the control sample indicates thesubject has a tissue proliferation-associated disorder.

In a further aspect, the invention includes a method of diagnosing atissue proliferation-associated disorder in a subject. The methodincludes providing a nucleic acid sample from the subject andidentifying at least a portion of the nucleotide sequence of a FGF-CXnucleic acid in the subject nucleic acid sample. The FGF-CX nucleotidesequence of the subject sample is then compared to a FGF-CX nucleotidesequence of a control sample. An alteration in the FGF-CX nucleotidesequence in the sample relative to the FGF-CX nucleotide sequence insaid control sample indicates the subject has a tissueproliferation-associated disorder.

In a still further aspect, the invention provides method of treating orpreventing or delaying a tissue proliferation-associated disorder. Themethod includes administering to a subject in which such treatment orprevention or delay is desired a FGF-CX nucleic acid, a FGF-CXpolypeptide, or a FGF-CX antibody in an amount sufficient to treat,prevent, or delay a tissue proliferation-associated disorder in thesubject.

The tissue proliferation-associated disorders diagnosed, treated,prevented or delayed using the FGF-CX nucleic acid molecules,polypeptides or antibodies can involve epithelial cells, e.g.,fibroblasts and keratinocytes in the anterior eye after surgery. Othertissue proliferation-associated disorder include, e.g., tumors,restenosis, psoriasis, Dupuytren's contracture, diabetic complications,Kaposi sarcoma, and rheumatoid arthritis.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representation of the nucleotide sequence (SEQ ID NO:1) andtranslated amino acid sequence (SEQ ID NO:2) of a novel FGF-CXpolynucleotide and protein of the invention.

FIG. 2 is a BLASTN alignment of the nucleic acid sequence of SEQ ID NO:1with a FGF-9-like Glia-Activating factor (GAF) sequence (SEQ ID NO:5).

FIG. 3 is a BLASTN alignment of the complementary strand of the nucleicacid sequence of SEQ ID NO:1 with three discontinuous segments (SEQ IDNOs:6-8 in panels A-C, respectively) of an extended genomic fragment ofhuman chromosome 8 (GenBank accession number AB020858).

FIG. 4 is a graphic representation of a hydropathy plot of the FGF-CXpolypeptide of SEQ ID NO:2, generated with a nineteen residue window.

FIG. 5 is a BLASTP alignment of the FGF-CX polypeptide sequence (SEQ IDNO:2) with a human FGF-9 (SEQ ID NO:9) indicating identical (“|”) andpositive (“+”) residues.

FIG. 6 is a BLASTX alignment of the FGF-CX polypeptide sequence (SEQ IDNO:2) with murine FGF-9 (SEQ ID NO:10) indicating identical (“|”) andpositive (“+”) residues.

FIG. 7 is a BLASTX alignment of the FGF-CX polypeptide sequence (SEQ IDNO:2) with rat FGF-9 (SEQ ID NO:11) indicating identical (“|”) andpositive (“+”) residues.

FIG. 8 is a BLASTX alignment of the FGF-CX polypeptide sequence (SEQ IDNO:2) with Xenopus XFGF-20 (SEQ ID NO:12) indicating identical (“|”) andpositive (“+”) residues.

FIG. 9 is a ClustalW Protein Sequence Alignment Analysis of fourvertebrate FGF-like proteins (SEQ ID NO:9-12) with the FGF-CX protein(SEQ ID NO:2) of the present invention. Black, gray and white representidentical, conserved and nonconserved residues in the alignment,respectively.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based in part on the discovery of novel FGF-CX nucleicacid sequence, which encode a polypeptide that is a member of thefibroblast growth factor family.

Included within the invention are FGF-CX nucleic acids, isolated nucleicacids that encode FGF-CX polypeptide or a portion thereof, FGF-CXpolypeptides, vectors containing these nucleic acids, host cellstransformed with the FGF-CX nucleic acids, anti-FGF-CX antibodies, andpharmaceutical compositions. Also disclosed are methods of making FGF-CXpolypeptides, as well as methods of screening, diagnosing, treatingconditions using these compounds, and methods of screenings compoundsthat modulate FGF-CX polypeptide activity. Table I below delineates thesequence descriptors that are used throughout the invention.

TABLE 1 SEQ ID NO SEQUENCE DESCRIPTOR 1 Human FGF-CX nucleotide sequence2 Human FGF-CX polypeptide sequence 3 FGF-CX forward primer 4 FGF-CXreverse primer 5 Glia Activating Factor (GAF) 6 Human genomic fragment -bp 15927-16214 7 Human genomic fragment - bp 7257-7511 8 Human genomicfragment - bp 9837-9942 9 Human FGF-9 10 Mouse FGF-9 11 Rat FGF-9 12Xenopus FGF-20 13 Human FGF-CX hydrophobic domain (aa 90-115)

The FGF-CX nucleic acids and polypeptides, as well as FGF-CX antibodies,as well as pharmacical compositions discussed herein, are useful, interalia, in treating tissue proliferation-associated disorders. Thesetissue proliferation-associated disorders can include epithelial cells,e.g., fibroblasts and keratinocytes in the anterior eye after surgery.Other tissue proliferation-associated disorder include, e.g., tumors,restenosis, psoriasis, Dupuytren's contracture, diabetic complications,Kaposi sacrcoma, and rheumatoid arthritis.

The present invention discloses a nucleotide sequence encoding a novelfibroblast growth factor designated fibroblast growth factor-CX (FGF-CX)herein (see FIG. 1; SEQ ID NO:1). This coding sequence was identified inhuman genomic DNA sequences. The DNA sequence has 633 bases that encodea polypeptide predicted to have 211 amino acid residues. The predictedmolecular weight of FGF-CX, based on the sequence shown in FIG. 1 andSEQ ID NO:2, is 23498.4 Da.

The FGF-CX nucleic acid sequence was used in a BLASTN search. The FGF-CXnucleotide sequence has a high similarity to murine fibroblast growthfactor 9 (FGF-9) (392 of 543 bases identical, or 72%; GenBank accessionnumber S82023) and to human DNA encoding glia activating factor (GAP)(385 of 554 bases identical, or 69%; GenBank accession number E05822,also termed FGF-9). In addition, FGF-CX was found to have a comparabledegree of identity (311 of 424 bases identical, or 73%) to a GAPsequence (SEQ ID NO:5) disclosed by Naruo et al. in Japanese Patent: JP1993301893 entitled “Glia-Activating Factor And Its Production” (seeFIG. 2).

An additional BLASTN search, shown in FIG. 3A-C, identified a humangenomic fragment of approximately 100,000 bp from chromosome 8 (GenBankaccession number AB020858, Homo sapiens genomic DNA of chromosome 8 p21.3-p22) with 3 widely separated sequences that, each individually,match a different portion of the contiguous sequence of the presentinvention almost perfectly. Specifically, the chromosomal sequence frombp 15927-16214 (SEQ ID NO:6) shown in FIG. 3A is 99% identical to thenoncoding FGF-CX strand from bp 1-289. The chromosomal sequence from bp7257-7511 (SEQ ID NO:7) shown in FIG. 3B is 98% identical to thenoncoding FGF-CX strand from bp 380-633. The chromosomal sequence frombp 9837-9942 (SEQ ID NO:8) shown in FIG. 3C is 100% identical to thenoncoding FGF-CX strand from bp 286-391. However, these genomicfragments shown in FIG. 3A-C differ from the present invention in thatthe genomic fragments (a) are separated from one another by severalthousand bases, (b) have a different order in the genomic sequencecompared to the order of the equivalent sequences in the presentlydisclosed nucleotide sequence, (c) represent the noncoding strand of thedisclosed invention, which is indicated in FIG. 3 by the reversednumbering of the FGF-CX nucleotides, and (d) are not associated oridentified in the GenBank disclosure in any way. Finally, even if theproper coding strand of the genomic sequence shown in FIG. 3A hadpreviously been identified, which it had not, and that strand had beenidentified as having an open reading frame, which it had not, thisportion of the genomic sequence has a one base deletion compared to theFGF-CX sequence of SEQ ID NO:1. See, FIG. 3A. Therefore, the predictedpolypeptide from the genomic sequence would be a different,frame-shifted, polypeptide from that point on compared to SEQ ID NO:2.

The polypeptide sequence in FIG. 1 (SEQ ID NO:2) is predicted by theprogram PSORT to have high probabilities for sorting through themembrane of the endoplasmic reticulum and of the microbody (peroxisome).In addition, although it does not have a predicted cleavable signalsequence at its N-terminus, the hydropathy plot in FIG. 4 shows FGF-CXhas a prominent hydrophobic segment at amino acid positions about 90 toabout 115 (SEQ ID NO:13). This single hydrophobic region is known to bea sorting signal in other members of the FGF family. According, apolypeptide that includes the amino acids of SEQ ID NO:13 is useful as asorting signal, allowing secretion through various cellular membranes,such as the endoplasmic reticulum, the Golgi membrane or the plasmamembrane.

A BLASTP alignment of the first 208 amino acids of the FGF-CXpolypeptide sequence (SEQ ID NO:2) with a human FGF-9 (SEQ ID NO:9) isshown in FIG. 5. See, SwissProt accession number P31371 forGlia-Activating Factor Precursor (GAF) (Fibroblast Growth Factor-9);Miyamoto et al. 1993 Mol. Cell. Biol. 13:4251-4259; and Naruo et al.1993 J. Biol Chem. 268:2857-2864. BLASTX alignments of the first 208amino acids of the FGF-CX polypeptide (SEQ ID NO:2, translated from SEQID NO:1) with the mouse FGF-9 (SEQ ID NO:10) and rat FGF-9 (SEQ IDNO:11) sequences are shown in FIGS. 6 and 7, respectively. See,SwissProt accession number P54130 for Glia-Activating Factor Precursor(GAF) (Fibroblast Growth Factor-9), Santos-Ocampo et al., 1996 J Biol.Chem. 271:1726-1731, for mouse FGF-9; and SwissProt accession numberP36364 Glia-Activating Factor Precursor (GAF) (Fibroblast GrowthFactor-9) (FGF-9), Miyamoto, 1993 Mol Cell. Biol. 13:4251-4259, for ratFGF-9. As indicated by the bars (“|”) in FIGS. 5-7, FGF-9 sequences ofall three species have 147 of 208 residues identical with FGF-CX (SEQ IDNO:2), for an overall sequence identity of 70%. In addition, 170 of 208residues are positive to the sequence of FGF-CX (SEQ ID NO:2), for anoverall percentage of positive residues of 81%. Positive residuesinclude those residues that are either identical (“|”) or have aconservative amino acid substitution (“+”) in the same relative positionof the compared sequences when aligned, see below.

The full length FGF-CX polypeptide (SEQ ID NO:2) was also aligned byBLASTX with Xenopus XFGF-20 (SEQ ID NO:12). As shown in FIG. 8, FGF-CXhas 170 of 211 (80%) identical residues, and 189 of 211 (89%) positiveresidues compared with Xenopus XFGF-20. Xenopus XFGF-20 was obtainedrecently from a cDNA library prepared at the tailbud stage using theproduct of degenerate PCR performed with primers based on mammalianFGF-9s as a probe. See, Koga et al., 1999 Biochem Biophys Res Commun261(3):756-765. The deduced 208 amino acid sequence of the XFGF-20 openreading frame contains a motif characteristic of the FGF family. XFGF-20has a 73.1% overall similarity to XFGF-9 but differs from XFGF-9 in itsamino-terminal region (33.3% similarity). This resembles the similarityseen for the presently disclosed SEQ ID NO:2 with respect to variousmammalian FGF-9 sequences, including human (see above). See, FIGS. 6-9.

XFGF-20 and XFGF-9 expression is distinct from each other. XFGF-20 mRNAis expressed in diploid cells, in embryos at and after the blastulastage, and specifically in the stomach and testis of adults; whereasXFGF-9 mRNA is expressed maternally in eggs and in many adult tissues.Koga et al., above. Correct expression of XFGF-20 during gastrulationappears to be required for the formation of normal head structures inXenopus laevis. When XFGF-20 mRNA was overexpressed in early embryos,gastrulation was abnormal and development of anterior structures wassuppressed. See, Koga et al., above. In such embryos, expression of theXbra transcript, among those tested, was suppressed during gastrulation,indicating that expression of the Xbra gene mediates XFGF-20 effects.See, Koga et al., above.

The present inventors believe that the results and observations providedby Koga et al., namely, the general correlation of expression of thegene in proliferating tissues including ova, testis, stomach, and manytissues in the maternal frog, suggests a role for XFGF-20 in themaintenance of tissues that normally undergo regeneration in afunctioning organism.

A ClustalW multiple protein alignment for the five vertebrate FGF-likeproteins discussed above, including the FGF-CX of the present invention,is shown in FIG. 9. The three mammalian proteins (SEQ ID NOs:9-11)resemble each other very closely but differ considerably from the FGF-CXprotein of the present invention (SEQ ID NO:2). Also, the XenopusXFGF-20 (SEQ ID NO:12) and the sequence of SEQ ID NO:2 resemble eachother more closely than those of FGF-9.

FGF-CX Nucleic Acids

The novel nucleic acids of the invention include those that encode aFGF-CX or FGF-CX-like protein. Among these nucleic acids is the nucleicacid whose sequence is provided in FIG. 1 and SEQ ID NO:1, or a fragmentthereof. Additionally, the invention includes mutant or variant nucleicacids of SEQ ID NO:1, or a fragment thereof, any of whose bases may bechanged from the corresponding base shown in FIG. 1 while still encodinga protein that maintains its FGF-CX-like activities and physiologicalfunctions. The invention further includes the complement of the nucleicacid sequence of SEQ ID NO:1, including fragments, derivatives, analogsand homolog thereof. Examples of the complementary strand of portions ofFGF-CX are shown in FIG. 3. The invention additionally includes nucleicacids or nucleic acid fragments, or complements thereto, whosestructures include chemical modifications.

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode FGF-CX proteins or biologically active portions thereof.Also included are nucleic acid fragments sufficient for use ashybridization probes to identify FGF-CX-encoding nucleic acids (e.g.,FGF-CX mRNA) and fragments for use as polymerase chain reaction (PCR)primers for the amplification or mutation of FGF-CX nucleic acidmolecules. As used herein, the term “nucleic acid molecule” is intendedto include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules(e.g., mRNA), analogs of the DNA or RNA generated using nucleotideanalogs, and derivatives, fragments and homologs thereof. The nucleicacid molecule can be single-stranded or double-stranded, but preferablyis double-stranded DNA.

“Probes” refer to nucleic acid sequences of variable length, preferablybetween at least about 10 nucleotides (nt), 100 nt, or as many as about,e.g., 6,000 nt, depending on use. Probes are used in the detection ofidentical, similar, or complementary nucleic acid sequences. Longerlength probes are usually obtained from a natural or recombinant source,are highly specific and much slower to hybridize than oligomers. Probesmay be single- or double-stranded and designed to have specificity inPCR, membrane-based hybridization technologies, or ELISA-liketechnologies.

An “isolated” nucleic acid molecule is one that is separated from othernucleic acid molecules that are present in the natural source of thenucleic acid. Examples of isolated nucleic acid molecules include, butare not limited to, recombinant DNA molecules contained in a vector,recombinant DNA molecules maintained in a heterologous host cell,partially or substantially purified nucleic acid molecules, andsynthetic DNA or RNA molecules. Preferably, an “isolated” nucleic acidis free of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated FGF-CX nucleic acidmolecule can contain less than about 50 kb, 25 kb, 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flankthe nucleic acid molecule in genomic DNA of the cell from which thenucleic acid is derived. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial or culture medium when produced by recombinant techniques, orof chemical precursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, or a complementof any of this nucleotide sequence, can be isolated using standardmolecular biology techniques and the sequence information providedherein. Using all or a portion of the nucleic acid sequence of SEQ IDNO:1 as a hybridization probe, FGF-CX nucleic acid sequences can beisolated using standard hybridization and cloning techniques (e.g., asdescribed in Sambrook et al., eds., MOLECULAR CLONING: A LABORATORYMANUAL 2^(nd) Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989; and Ausubel, et al., eds., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993.)

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to FGF-CX nucleotidesequences can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

As used herein, the term “oligonucleotide” refers to a series of linkednucleotide residues, which oligonucleotide has a sufficient number ofnucleotide bases to be used in a PCR reaction. A short oligonucleotidesequence may be based on, or designed from, a genomic or cDNA sequenceand is used to amplify, confirm, or reveal the presence of an identical,similar or complementary DNA or RNA in a particular cell or tissue.Oligonucleotides comprise portions of a nucleic acid sequence havingabout 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 ntin length. In one embodiment, an oligonucleotide comprising a nucleicacid molecule less than 100 nt in length would further comprise at lease6 contiguous nucleotides of SEQ ID NO:1, or a complement thereof.Oligonucleotides may be chemically synthesized and may be used asprobes.

In another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule that is a complement of thenucleotide sequence shown in SEQ ID NO:1. In another embodiment, anisolated nucleic acid molecule of the invention comprises a nucleic acidmolecule that is a complement of the nucleotide sequence shown in SEQ IDNO:1, or a portion of this nucleotide sequence. A nucleic acid moleculethat is complementary to the nucleotide sequence shown in SEQ ID NO:1 isone that is sufficiently complementary to the nucleotide sequence shownin SEQ ID NO:1 that it can hydrogen bond with little or no mismatches tothe nucleotide sequence shown in SEQ ID NO:1, thereby forming a stableduplex.

As used herein, the term “complementary” refers to Watson-Crick orHoogsteen base pairing between nucleotides units of a nucleic acidmolecule, and the term “binding” means the physical or chemicalinteraction between two polypeptides or compounds or associatedpolypeptides or compounds or combinations thereof. Binding includesionic, non-ionic, Von der Waals, hydrophobic interactions, etc. Aphysical interaction can be either direct or indirect. Indirectinteractions may be through or due to the effects of another polypeptideor compound. Direct binding refers to interactions that do not takeplace through, or due to, the effect of another polypeptide or compound,but instead are without other substantial chemical intermediates.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO:1, e.g., a fragmentthat can be used as a probe or primer, or a fragment encoding abiologically active portion of FGF-CX. Fragments provided herein aredefined as sequences of at least 6 (contiguous) nucleic acids or atleast 4 (contiguous) amino acids, a length sufficient to allow forspecific hybridization in the case of nucleic acids or for specificrecognition of an epitope in the case of amino acids, respectively, andare at most some portion less than a full length sequence. Fragments maybe derived from any contiguous portion of a nucleic acid or amino acidsequence of choice. Derivatives are nucleic acid sequences or amino acidsequences formed from the native compounds either directly or bymodification or partial substitution. Analogs are nucleic acid sequencesor amino acid sequences that have a structure similar to, but notidentical to, the native compound but differs from it in respect tocertain components or side chains. Analogs may be synthetic or from adifferent evolutionary origin and may have a similar or oppositemetabolic activity compared to wild type.

Derivatives and analogs may be full length or other than full length, ifthe derivative or analog contains a modified nucleic acid or amino acid,as described below. Derivatives or analogs of the nucleic acids orproteins of the invention include, but are not limited to, moleculescomprising regions that are substantially homologous to the nucleicacids or proteins of the invention, in various embodiments, by at leastabout 70%, 80%, 85%, 90%, 95%, 98%, or even 99% identity (with apreferred identity of 80-99%) over a nucleic acid or amino acid sequenceof identical size or when compared to an aligned sequence in which thealignment is done by a computer homology program known in the art, orwhose encoding nucleic acid is capable of hybridizing to the complementof a sequence encoding the aforementioned proteins under stringent,moderately stringent, or low stringent conditions. See e.g. Ausubel, etal., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NewYork, N.Y., 1993, and below. An exemplary program is the Gap program(Wisconsin Sequence Analysis Package, Version 8 for UNIX, GeneticsComputer Group, University Research Park, Madison, Wis.) using thedefault settings, which uses the algorithm of Smith and Waterman (Adv.Appl. Math., 1981, 2: 482-489, which is incorporated herein by referencein its entirety).

A “homologous nucleic acid sequence” or “homologous amino acidsequence,” or variations thereof, refer to sequences characterized by ahomology at the nucleotide level or amino acid level as discussed above.Homologous nucleotide sequences encode those sequences coding forisoforms of FGF-CX polypeptide. Isoforms can be expressed in differenttissues of the same organism as a result of, for example, alternativesplicing of RNA. Alternatively, isoforms can be encoded by differentgenes. In the present invention, homologous nucleotide sequences includenucleotide sequences encoding for a FGF-CX polypeptide of species otherthan humans, including, but not limited to, mammals, and thus caninclude, e.g., mouse, rat, rabbit, dog, cat cow, horse, and otherorganisms. Homologous nucleotide sequences also include, but are notlimited to, naturally occurring allelic variations and mutations of thenucleotide sequences set forth herein. A homologous nucleotide sequencedoes not, however, include the nucleotide sequence encoding human FGF-CXprotein. Homologous nucleic acid sequences include those nucleic acidsequences that encode conservative amino acid substitutions (see below)in SEQ ID NO:2, as well as a polypeptide having FGF-CX activity.Biological activities of the FGF-CX proteins are described below. Ahomologous amino acid sequence does not encode the amino acid sequenceof a human FGF-CX polypeptide.

The nucleotide sequence determined from the cloning of the human FGF-CXgene allows for the generation of probes and primers designed for use inidentifying and/or cloning FGF-CX homologues in other cell types, e.g.,from other tissues, as well as FGF-CX homologues from other mammals. Theprobe/primer typically comprises a substantially purifiedoligonucleotide. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 or moreconsecutive sense strand nucleotide sequence of SEQ ID NO:1; or ananti-sense strand nucleotide sequence of SEQ ID NO:1; or of a naturallyoccurring mutant of SEQ ID NO:1.

Probes based on the human FGF-CX nucleotide sequence can be used todetect transcripts or genomic sequences encoding the same or homologousproteins. In various embodiments, the probe further comprises a labelgroup attached thereto, e.g., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a diagnostic test kit for identifying cells ortissue which misexpress a FGF-CX protein, such as by measuring a levelof a FGF-CX-encoding nucleic acid in a sample of cells from a subjecte.g., detecting FGF-CX mRNA levels or determining whether a genomicFGF-CX gene has been mutated or deleted.

“A polypeptide having a biologically active portion of FGF-CX” refers topolypeptides exhibiting activity similar, but not necessarily identicalto, an activity of a polypeptide of the present invention, includingmature forms, as measured in a particular biological assay, with orwithout dose dependency. A nucleic acid fragment encoding a“biologically active portion of FGF-CX” can be prepared by isolating aportion of SEQ ID NO:1, that encodes a polypeptide having a FGF-CXbiological activity (biological activities of the FGF-CX proteins aredescribed below), expressing the encoded portion of FGF-CX protein(e.g., by recombinant expression in vitro) and assessing the activity ofthe encoded portion of FGF-CX. For example, a nucleic acid fragmentencoding a biologically active portion of FGF-CX can optionally includean ATP-binding domain. In another embodiment, a nucleic acid fragmentencoding a biologically active portion of FGF-CX includes one or moreregions.

FGF-CX Variants

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequences shown in FIG. 1 due to degeneracy of thegenetic code. These nucleic acids thus encode the same FGF-CX protein asthat encoded by the nucleotide sequence shown in SEQ ID NO:1. In anotherembodiment, an isolated nucleic acid molecule of the invention has anucleotide sequence encoding a protein having an amino acid sequenceshown in SEQ ID NO:2.

In addition to the human FGF-CX nucleotide sequence shown in SEQ IDNO:1, it will be appreciated by those skilled in the art that DNAsequence polymorphisms that lead to changes in the amino acid sequencesof FGF-CX may exist within a population (e.g., the human population).Such genetic polymorphism in the FGF-CX gene may exist among individualswithin a population due to natural allelic variation. As used herein,the terms “gene” and “recombinant gene” refer to nucleic acid moleculescomprising an open reading frame encoding a FGF-CX protein, preferably amammalian FGF-CX protein. Such natural allelic variations can typicallyresult in 1-5% variance in the nucleotide sequence of the FGF-CX gene.Any and all such nucleotide variations and resulting amino acidpolymorphisms in FGF-CX that are the result of natural allelic variationand that do not alter the functional activity of FGF-CX are intended tobe within the scope of the invention.

Moreover, nucleic acid molecules encoding FGF-CX proteins from otherspecies, and thus that have a nucleotide sequence that differs from thehuman sequence of SEQ ID NO:1, are intended to be within the scope ofthe invention. Nucleic acid molecules corresponding to natural allelicvariants and homologues of the FGF-CX cDNAs of the invention can beisolated based on their homology to the human FGF-CX nucleic acidsdisclosed herein using the human cDNAs, or a portion thereof, as ahybridization probe according to standard hybridization techniques understringent hybridization conditions. For example, a soluble human FGF-CXcDNA can be isolated based on its homology to human membrane-boundFGF-CX. Likewise, a membrane-bound human FGF-CX cDNA can be isolatedbased on its homology to soluble human FGF-CX.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 6 nucleotides in length and. hybridizes understringent conditions to the nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1. In another embodiment, the nucleicacid is at least 10, 25, 50, 100, 250, 500 or 750 nucleotides in length.In another embodiment, an isolated nucleic acid molecule of theinvention hybridizes to the coding region. As used herein, the term“hybridizes under stringent conditions” is intended to describeconditions for hybridization and washing under which nucleotidesequences at least 60% homologous to each other typically remainhybridized to each other.

Homologs (i.e., nucleic acids encoding FGF-CX proteins derived fromspecies other than human) or other related sequences (e.g., paralogs)can be obtained by low, moderate or high stringency hybridization withall or a portion of the particular human sequence as a probe usingmethods well known in the art for nucleic acid hybridization andcloning.

As used herein, the phrase “stringent hybridization conditions” refersto conditions under which a probe, primer or oligonucleotide willhybridize to its target sequence, but to no other sequences. Stringentconditions are sequence-dependent and will be different in differentcircumstances. Longer sequences hybridize specifically at highertemperatures than shorter sequences. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (Tm) forthe specific sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength, pH and nucleic acidconcentration) at which 50% of the probes complementary to the targetsequence hybridize to the target sequence at equilibrium. Since thetarget sequences are generally present at excess, at Tm, 50% of theprobes are occupied at equilibrium. Typically, stringent conditions willbe those in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0to 8.3 and the temperature is at least about 30° C. for short probes,primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about60° C. for longer probes, primers and oligonucleotides. Stringentconditions may also be achieved with the addition of destabilizingagents, such as formamide.

Stringent conditions are known to those skilled in the art and can befound in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequencesat least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous toeach other typically remain hybridized to each other. A non-limitingexample of stringent hybridization conditions is hybridization in a highsalt buffer comprising 6×SSC, 50 mM Tris-HCI (pH 7.5), 1 mM EDTA, 0.02%PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNAat 65° C. This hybridization is followed by one or more washes in0.2×SSC, 0.01% BSA at 50° C. An isolated nucleic acid molecule of theinvention that hybridizes under stringent conditions to the sequence ofSEQ ID NO:1 corresponds to a naturally occurring nucleic acid molecule.As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature (e.g., encodes a natural protein).

In a second embodiment, a nucleic acid sequence that is hybridizable tothe nucleic acid molecule comprising the nucleotide sequence of SEQ IDNO:1, or fragments, analogs or derivatives thereof, under conditions ofmoderate stringency is provided. A non-limiting example of moderatestringency hybridization conditions are hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNAat 55° C., followed by one or more washes in 1×SSC, 0.1% SDS at 37° C.Other conditions of moderate stringency that may be used are well knownin the art. See, e.g., Ausubel et al. (eds.), 1993, CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENETRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.

In a third embodiment, a nucleic acid that is hybridizable to thenucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1,or fragments, analogs or derivatives thereof, under conditions of lowstringency, is provided. A non-limiting example of low stringencyhybridization conditions are hybridization in 35% formamide, 5×SSC, 50mM Tris-HCI (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100mg/mn denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C., followed by one or more washes in 2×SSC, 25 mM Tris-HCI (pH 7.4), 5mM EDTA, and 0.1% SDS at 50° C. Other conditions of low stringency thatmay be used are well known in the art (e.g., as employed forcross-species hybridizations). See, e.g., Ausubel et al. (eds.), 1993,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, andKriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL,Stockton Press, NY; Shilo and Weinberg, 1981, Proc Natl Acad Sci USA 78:6789-6792.

Conservative Mutations

In addition to naturally-occurring allelic variants of the FGF-CXsequence that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into thenucleotide sequence of SEQ ID NO:1, thereby leading to changes in theamino acid sequence of the encoded FGF-CX protein, without altering thefunctional ability of the FGF-CX protein. For example, nucleotidesubstitutions leading to amino acid substitutions at “non-essential”amino acid residues can be made in the sequence of SEQ ID NO:1. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequence of FGF-CX without altering the biologicalactivity, whereas an “essential” amino acid residue is required forbiological activity. For example, amino acid residues that are conservedamong the FGF-CX proteins of the present invention, are predicted to beparticularly unamenable to alteration.

In addition, amino acid residues that are conserved among FGF familymembers, as indicated by the alignment presented as FIG. 9, arepredicted to be less amenable to alteration. For example, FGF-CXproteins of the present invention can contain at least one domain thatis a typically conserved region in FGF family members, i.e., FGF-9 andXFGF-20 proteins, and FGF-CX homologs. As such, these conserved domainsare not likely to be amenable to mutation. Other amino acid residues,however, (e.g., those that are not conserved or only semi-conservedamong members of the FGF proteins) may not be as essential for activityand thus are more likely to be amenable to alteration.

Another aspect of the invention pertains to nucleic acid moleculesencoding FGF-CX proteins that contain changes in amino acid residuesthat are not essential for activity. Such FGF-CX proteins differ inamino acid sequence from SEQ ID NO:2, yet retain biological activity. Inone embodiment, the isolated nucleic acid molecule comprises anucleotide sequence encoding a protein, wherein the protein comprises anamino acid sequence at least about 75% homologous to the amino acidsequence of SEQ ID NO:2. Preferably, the protein encoded by the nucleicacid is at least about 80% homologous to SEQ ID NO:2, more preferably atleast about 90%, 95%, 98%, and most preferably at least about 99%homologous to SEQ ID NO:2.

An isolated nucleic acid molecule encoding a FGF-CX protein homologousto the protein of SEQ ID NO:2 can be created by introducing one or morenucleotide substitutions, additions or deletions into the nucleotidesequence of SEQ ID NO:1, such that one or more amino acid substitutions,additions or deletions are introduced into the encoded protein.

Mutations can be introduced into SEQ ID NO:1 by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.Preferably, conservative amino acid substitutions are made at one ormore predicted non-essential amino acid residues. A “conservative minoacid substitution” is one in which the amino acid residue is replacedwith an amino acid residue having a similar side chain. Families ofamino acid residues having similar side chains have been defined in theart. These families include amino acids with basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted nonessentialamino acid residue in FGF-CX is replaced with another amino acid residuefrom the same side chain family. Alternatively, in another embodiment,mutations can be introduced randomly along all or part of a FGF-CXcoding sequence, such as by saturation mutagenesis, and the resultantmutants can be screened for FGF-CX biological activity to identifymutants that retain activity. Following mutagenesis of SEQ ID NO:1, theencoded protein can be expressed by any recombinant technology known inthe art and the activity of the protein can be determined.

In one embodiment, a mutant FGF-CX protein can be assayed for (1) theability to form protein:protein interactions with other FGF-CX proteins,other cell-surface proteins, or biologically active portions thereof,(2) complex formation between a mutant FGF-CX protein and a FGF-CXreceptor; (3) the ability of a mutant FGF-CX protein to bind to anintracellular target protein or biologically active portion thereof;(e.g., avidin proteins); (4) the ability to bind BRA protein; or (5) theability to specifically bind an anti-FGF-CX protein antibody.

Antisense

Another aspect of the invention pertains to isolated antisense nucleicacid molecules that are hybridizable to or complementary to the nucleicacid molecule comprising the nucleotide sequence of SEQ ID NO:1, orfragments, analogs or derivatives thereof. An “antisense” nucleic acidcomprises a nucleotide sequence that is complementary to a “sense”nucleic acid encoding a protein, e.g., complementary to the codingstrand of a double-stranded cDNA molecule or complementary to an mRNAsequence. In specific aspects, antisense nucleic acid molecules areprovided that comprise a sequence complementary to at least about 10,25, 50, 100, 250 or 500 nucleotides or an entire FGF-CX coding strand,or to only a portion thereof. Nucleic acid molecules encoding fragments,homologs, derivatives and analogs of a FGF-CX protein of SEQ ID NO:2 orantisense nucleic acids complementary to a FGF-CX nucleic acid sequenceof SEQ ID NO:1 are additionally provided.

In one embodiment, an antisense nucleic acid molecule is antisense to a“coding region” of the coding strand of a nucleotide sequence encodingFGF-CX. The term “coding region” refers to the region of the nucleotidesequence comprising codons which are translated into amino acid residues(e.g., the protein coding region of human FGF-CX corresponds to SEQ IDNO:2). In another embodiment, the antisense nucleic acid molecule isantisense to a “noncoding region” of the coding strand of a nucleotidesequence encoding FGF-CX. The term “noncoding region” refers to 5′ and3′ sequences which flank the coding region that are not translated intoamino acids (i.e., also referred to as 5′ and 3′ untranslated regions).

Given the coding strand sequences encoding FGF-CX disclosed herein(e.g., SEQ ID NO:1), antisense nucleic acids of the invention can bedesigned according to the rules of Watson and Crick or Hoogsteen basepairing. The antisense nucleic acid molecule can be complementary to theentire coding region of FGF-CX mRNA, but more preferably is anoligonucleotide that is antisense to only a portion of the coding ornoncoding region of FGF-CX mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of FGF-CX mRNA. An antisense oligonucleotide canbe, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50nucleotides in length. An antisense nucleic acid of the invention can beconstructed using chemical synthesis or enzymatic ligation reactionsusing procedures known in the art. For example, an antisense nucleicacid (e.g., an antisense oligonucleotide) can be chemically synthesizedusing naturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used.

Examples of modified nucleotides that can be used to generate theantisense nucleic acid include: 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a FGF-CXprotein to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule that binds toDNA duplexes, through specific interactions in the major groove of thedouble helix. An example of a route of administration of antisensenucleic acid molecules of the invention includes direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, for systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies that bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of antisense molecules,vector constructs in which the antisense nucleic acid molecule is placedunder the control of a strong pol II or pol III promoter are preferred.

In-yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids Res 15: 6625-6641). Theantisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett215: 327-330).

Ribozymes and PNA Moieties

Such modifications include, by way of nonlimiting example, modifiedbases, and nucleic acids whose sugar phosphate backbones are modified orderivatized. These modifications are carried out at least in part toenhance the chemical stability of the modified nucleic acid, such thatthey may be used, for example, as antisense binding nucleic acids intherapeutic applications in a subject.

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity that are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haselhoff andGerlach (1988) Nature 334:585-591)) can be used to catalytically cleaveFGF-CX mRNA transcripts to thereby inhibit translation of FGF-CX mRNA. Aribozyme having specificity for a FGF-CX-encoding nucleic acid can bedesigned based upon the nucleotide sequence of a FGF-CX DNA disclosedherein (i.e., SEQ ID NO:1). For example, a derivative of a TetrahymenaL-19 IVS RNA can be constructed in which the nucleotide sequence of theactive site is complementary to the nucleotide sequence to be cleaved ina FGF-CX-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071;and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, FGF-CX mRNA canbe used to select a catalytic RNA having a specific ribonucleaseactivity from a pool of RNA molecules. See, e.g., Bartel et al., (1993)Science 261:1411-1418.

Alternatively, FGF-CX gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of theFGF-CX (e.g., the FGF-CX promoter and/or enhancers) to form triplehelical structures that prevent transcription of the FGF-CX gene intarget cells. See generally, Helene. (1991) Anticancer Drug Des. 6:569-84; Helene. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher(1992) Bioassays 14: 807-15.

In various embodiments, the nucleic acids of FGF-CX can be modified atthe base moiety, sugar moiety or phosphate backbone to improve, e.g.,the stability, hybridization, or solubility of the molecule. Forexample, the deoxyribose phosphate backbone of the nucleic acids can bemodified to generate peptide nucleic acids (see Hyrup et al. (1996)Bioorg Med Chem 4: 5-23). As used herein, the terms “peptide nucleicacids” or “PNAs” refer to nucleic acid mimics, e.g. DNA mimics, in whichthe deoxyribose phosphate backbone is replaced by a pseudopeptidebackbone and only the four natural nucleobases are retained. The neutralbackbone of PNAs has been shown to allow for specific hybridization toDNA and RNA under conditions of low ionic strength. The synthesis of PNAoligomers can be performed using standard solid phase peptide synthesisprotocols as described in Hyrup et al. (1996) above; Perry-O'Keefe etal. (1996) PNAS 93: 14670-675.

PNAs of FGF-CX can be used in therapeutic and diagnostic applications.For example, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, e.g., inducingtranscription or translation arrest or inhibiting replication. PNAs ofFGF-CX can also be used, e.g., in the analysis of single base pairmutations in a gene by, e.g., PNA directed PCR clamping; as artificialrestriction enzymes when used in combination with other enzymes, e.g.,S1 nucleases (Hyrup B. (1996) above); or as probes or primers for DNAsequence and hybridization (Hyrup et al. (1996), above; Perry-O'Keefe(1996), above).

In another embodiment, PNAs of FGF-CX can be modified, e.g., to enhancetheir stability or cellular uptake, by attaching lipophilic or otherhelper groups to PNA, by the formation of PNA-DNA chimeras, or by theuse of liposomes or other techniques of drug delivery known in the art.For example, PNA-DNA chimeras of FGF-CX can be generated that maycombine the advantageous properties of PNA and DNA. Such chimeras allowDNA recognition enzymes, e.g., RNase H and DNA polymerases, to interactwith the DNA portion while the PNA portion would provide high bindingaffinity and specificity. PNA-DNA chimeras can be linked using linkersof appropriate lengths selected in terms of base stacking, number ofbonds between the nucleobases, and orientation (Hyrup (1996) above). Thesynthesis of PNA-DNA chimeras can be performed as described in Hyrup(1996) above and Finn et al. (1996) Nucl Acids Res 24: 3357-63. Forexample, a DNA chain can be synthesized on a solid support usingstandard phosphoramidite coupling chemistry, and modified nucleosideanalogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite, can be used between the PNA and the 5′ end of DNA (Maget al. (1989) Nucl Acid Res 17: 5973-88). PNA monomers are then coupledin a stepwise manner to produce a chimeric molecule with a 5′ PNAsegment and a 3′ DNA segment (Finn et al. (1996) above). Alternatively,chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNAsegment. See, Petersen et al. (1975) Bioorg Med Chem Lett 5: 1119-11124.

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A.86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652;PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g.,PCT Publication No. W089/10134). In addition, oligonucleotides can bemodified with hybridization triggered cleavage agents (See, e.g., Krolet al., 1988, BioTechniques 6:958-976) or intercalating agents. (See,e.g., Zon, 1988, Pharm. Res. 5: 539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,a hybridization triggered cross-linking agent, a transport agent, ahybridization-triggered cleavage agent, etc.

FGF-CX Polypeptides

The novel protein of the invention includes the FGF-CX-like proteinwhose sequence is provided in FIG. 1 (SEQ ID NO:2). The invention alsoincludes a mutant or variant protein any of whose residues may bechanged from the corresponding residue shown in FIG. 1 while stillencoding a protein that maintains its FGF-CX-like activities andphysiological functions, or a functional fragment thereof. In the mutantor variant protein, up to 20% or more of the residues may be so changed.

In general, an FGF-CX-like variant that preserves FGF-CX-like functionincludes any variant in which residues at a particular position in thesequence have been substituted by other amino acids, and further includethe possibility of inserting an additional residue or residues betweentwo residues of the parent protein as well as the possibility ofdeleting one or more residues from the parent sequence. Any amino acidsubstitution, insertion, or deletion is encompassed by the invention. Infavorable circumstances, the substitution is a conservative substitutionas defined above. Furthermore, without limiting the scope of theinvention, the following positions in Table 2 (using the numberingprovided in SEQ ID NO:2) may be substituted as indicated, such that amutant or variant protein may include one or more than one of thesubstitutions indicated. The suggested substitutions do not limit therange of possible substitutions that may be made at a given position.

TABLE 2 Position Possible Substitution 6: Glu to Asp 9: Gly to Ser, Thr,or Asn 10: Phe to Tyr 11: Leu to Phe or Ile 15: Glu to Asp 16: Gly toAla 17: Leu to Ile or Val 19: Gln may be deleted 21: Val to Phe or Ile31: Gly to Lys, Arg, Ser, or Ala 33: Arg to Lys or Ser 35: Pro to Leu orVal 38: Gly to Asn or Ser 39: Glu to Asp 40: Arg to Lys, His, or Pro 42:Ser to Thr, Ala, or Gly 43: Ala to Gln, Asn, or Ser 48: Ala to Ser orGly 51: Gly to Ala 53: Gly to Ala or deleted 54: Ala to Gly, Val, ordeleted 55: Ala to Ser or Thr 56: Gln to Asp, Glu, or Asn 58: Ala toSer, Thr, Asn, Gln, Asp, or Glu 61: His to Gln, Asn, Lys, or Arg 78: Glnto Asn, Glu, or Asp 80: Leu to Phe or Ile 82: Asp to Glu, Asn, or Gln84: Ser to Asn, Thr, or Gln 85: Val to Ile 90: Gln to Asn or Lys 103:Val to Ile 115: Ser to Thr 123: Asp to Glu 128: Tyr to Phe 135: Ser toThr, Gln, or Asn 138: Ile to Val or Leu 155: Ile to Leu 159: Gly to Valor Ala 161: Thr to Ser 166: Phe to Tyr 177: Asp to Glu 181: Ser to Alaor Thr 198: Glu to Asp 199: Arg to Lys 207: Leu to Ile or Val 209: Metto any residue 211: Thr to Ser

One aspect of the invention pertains to isolated FGF-CX proteins, andbiologically active portions thereof, or derivatives, fragments, analogsor homologs thereof. Also provided are polypeptide fragments suitablefor use as inmunogens to raise anti-FGF-CX antibodies. In oneembodiment, native FGF-CX proteins can be isolated from cells or tissuesources by an appropriate purification scheme using standard proteinpurification techniques. In another embodiment, FGF-CX proteins areproduced by recombinant DNA techniques. Alternative to recombinantexpression, a FGF-CX protein or polypeptide can be synthesizedchemically using standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theFGF-CX protein is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations ofFGF-CX protein in which the protein is separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. In one embodiment, the language “substantially free ofcellular material” includes preparations of FGF-CX protein having lessthan about 30% (by dry weight) of non-FGF-CX protein (also referred toherein as a “contaminating protein”), more preferably less than about20% of non-FGF-CX protein, still more preferably less than about 10% ofnon-FGF-CX protein, and most preferably less than about 5% non-FGF-CXprotein. When the FGF-CX protein or biologically active portion thereofis recombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of FGF-CX protein in which the proteinis separated from chemical precursors or other chemicals that areinvolved in the synthesis of the protein. In one embodiment, thelanguage “substantially free of chemical precursors or other chemicals”includes preparations of FGF-CX protein having less than about 30% (bydry weight) of chemical precursors or non-FGF-CX chemicals, morepreferably less than about 20% chemical precursors or non-FGF-CXchemicals, still more preferably less than about 10% chemical precursorsor non-FGF-CX chemicals, and most preferably less than about 5% chemicalprecursors or non-FGF-CX chemicals.

Biologically active portions of a FGF-CX protein include peptidescomprising amino acid sequences sufficiently homologous to or derivedfrom the amino acid sequence of the FGF-CX protein, e.g., the amino acidsequence shown in SEQ ID NO:2 that include fewer amino acids than thefull length FGF-CX proteins, and exhibit at least one activity of aFGF-CX protein. Typically, biologically active portions comprise adomain or motif with at least one activity of the FGF-CX protein. Abiologically active portion of a FGF-CX protein can be a polypeptidewhich is, for example, 10, 25, 50, 100 or more amino acids in length.

A biologically active portion of a FGF-CX protein of the presentinvention may contain at least one of the above-identified domainsconserved between the FGF family of proteins. Moreover, otherbiologically active portions, in which other regions of the protein aredeleted, can be prepared by recombinant techniques and evaluated for oneor more of the functional activities of a native FGF-CX protein.

In an embodiment, the FGF-CX protein has an amino acid sequence shown inSEQ ID NO:2 In other embodiments, the FGF-CX protein is substantiallyhomologous to SEQ ID NO:2 and retains the functional activity of theprotein of SEQ ID NO:2, yet differs in amino acid sequence due tonatural allelic variation or mutagenesis, as described in detail below.Accordingly, in another embodiment, the FGF-CX protein is a protein thatcomprises an amino acid sequence at least about 45% homologous to theamino acid sequence of SEQ ID NO:2 and retains the functional activityof the FGF-CX proteins of SEQ ID NO:2.

Determining Homology Between Two or More Sequences

To determine the percent homology of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in either of the sequences being comparedfor optimal alignment between the sequences). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules arehomologous at that position (i.e., as used herein amino acid or nucleicacid “homology” is equivalent to amino acid or nucleic acid “identity”).

The nucleic acid sequence homology may be determined as the degree ofidentity between two sequences. The homology may be determined usingcomputer programs known in the art, such as GAP software provided in theGCG program package. See, Needleman and Wunsch 1970 J Mol Biol 48:443-453. Using GCG GAP software with the following settings for nucleicacid sequence comparison: GAP creation penalty of 5.0 and GAP extensionpenalty of 0.3, the coding region of the analogous nucleic acidsequences referred to above exhibits a degree of identity preferably ofat least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS(encoding) part of the DNA sequence shown in SEQ ID NO:1.

The term “sequence identity” refers to the degree to which twopolynucleotide or polypeptide sequences are identical on aresidue-by-residue basis over a particular region of comparison. Theterm “percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over that region of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I, in the case of nucleic acids) occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the region ofcomparison (i.e., the window size), and multiplying the result by 100 toyield the percentage of sequence identity. The term “substantialidentity” as used herein denotes a characteristic of a polynucleotidesequence, wherein the polynucleotide comprises a sequence that has atleast 80 percent sequence identity, preferably at least 85 percentidentity and often 90 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison region. The term “percentage of positive residues” iscalculated by comparing two optimally aligned sequences over that regionof comparison, determining the number of positions at which theidentical and conservative amino acid substitutions, as defined above,occur in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the region of comparison (i.e., the window size), andmultiplying the result by 100 to yield the percentage of positiveresidues.

Chimeric and Fusion Proteins

The invention also provides FGF-CX chimeric or fusion proteins. As usedherein, a FGF-CX “chimeric protein” or “fusion protein” comprises aFGF-CX polypeptide operatively linked to a non-FGF-CX polypeptide. A“FGF-CX polypeptide” refers to a polypeptide having an amino acidsequence corresponding to FGF-CX, whereas a “non-FGF-CX polypeptide”refers to a polypeptide having an amino acid sequence corresponding to aprotein that is not substantially homologous to the FGF-CX protein,e.g., a protein that is different from the FGF-CX protein and that isderived from the same or a different organism. Within a FGF-CX fuisionprotein the FGF-CX polypeptide can correspond to all or a portion of aFGF-CX protein. In one embodiment, a FGF-CX fusion protein comprises atleast one biologically active portion of a FGF-CX protein. In anotherembodiment, a FGF-CX fusion protein comprises at least two biologicallyactive portions of a FGF-CX protein. Within the fusion protein, the term“operatively linked” is intended to indicate that the FGF-CX polypeptideand the non-FGF-CX polypeptide are fused in-frame to each other. Thenon-FGF-CX polypeptide can be fused to the N-terminus or C-terminus ofthe FGF-CX polypeptide.

For example, in one embodiment a FGF-CX fusion protein comprises aFGF-CX polypeptide operably linked to the extracellular domain of asecond protein. Such fusion proteins can be further utilized inscreening assays for compounds that modulate FGF-CX activity (suchassays are described in detail below).

In another embodiment, the fusion protein is a GST-FGF-CX fusion proteinin which the FGF-CX sequences are fused to the C-terminus of the GST(i.e., glutathione S-transferase) sequences. Such fusion proteins canfacilitate the purification of recombinant FGF-CX.

In yet another embodiment, the fusion protein is a FGF-CX proteincontaining a heterologous signal sequence at its N-terminus. Forexample, the native FGF-CX signal sequence (i.e., amino acids 1 to 20 ofSEQ ID NO:2 ) can be removed and replaced with a signal sequence fromanother protein. In certain host cells (e.g., mammalian host cells),expression and/or secretion of FGF-CX can be increased through use of aheterologous signal sequence.

In another embodiment, the fusion protein is a FGF-CX-immunoglobulinfusion protein in which the FGF-CX sequences comprising one or moredomains are fused to sequences derived from a member of theimmunoglobulin protein family. The FGF-CX-immunoglobulin fusion proteinsof the invention can be incorporated into pharmaceutical compositionsand administered to a subject to inhibit an interaction between a FGF-CXligand and a FGF-CX protein on the surface of a cell, to therebysuppress FGF-CX-mediated signal transduction in vivo. In one nonlimitingexample, a contemplated FGF-CX ligand of the invention is the FGF-CXreceptor. The FGF-CX-immunoglobulin fusion proteins can be used toaffect the bioavailability of a FGF-CX cognate ligand. Inhibition of theFGF-CX ligand/FGF-CX interaction may be useful therapeutically for boththe treatment of proliferative and differentiative disorders, as well asmodulating (e.g., promoting or inhibiting) cell survival. Moreover, theFGF-CX-immunoglobulin fusion proteins of the invention can be used asimmunogens to produce anti-FGF-CX antibodies in a subject, to purifyFGF-CX ligands, and in screening assays to identify molecules thatinhibit the interaction of FGF-CX with a FGF-CX ligand.

A FGF-CX chimeric or fusion protein of the invention can be produced bystandard recombinant DNA techniques. For example, DNA fragments codingfor the different polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, e.g., by employing blunt-endedor stagger-ended termiini for ligation, restriction enzyme digestion toprovide for appropriate termini, filling-in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers that give rise to complementaryoverhangs between two consecutive gene fragments that can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULARBIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). A FGF-CX-encoding nucleic acid can be cloned into such anexpression vector such that the fusion moiety is linked in-frame to theFGF-CX protein.

FGF-CX Agonists and Antagonists

The present invention also pertains to variants of the FGF-CX proteinsthat function as either FGF-CX agonists (mimetics) or as FGF-CXantagonists. Variants of the FGF-CX protein can be generated bymutagenesis, e.g., discrete point mutation or truncation of the FGF-CXprotein. An agonist of the FGF-CX protein can retain substantially thesame, or a subset of, the biological activities of the naturallyoccurring form of the FGF-CX protein. An antagonist of the FGF-CXprotein can inhibit one or more of the activities of the naturallyoccurring form of the FGF-CX protein by, for example, competitivelybinding to a downstream or upstream member of a cellular signalingcascade which includes the FGF-CX protein. Thus, specific biologicaleffects can be elicited by treatment with a variant of limited function.In one embodiment, treatment of a subject with a variant having a subsetof the biological activities of the naturally occurring form of theprotein has fewer side effects in a subject relative to treatment withthe naturally occurring form of the FGF-CX proteins.

Variants of the FGF-CX protein that function as either FGF-CX agonists(mimetics) or as FGF-CX antagonists can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, of theFGF-CX protein for FGF-CX protein agonist or antagonist activity. In oneembodiment, a variegated library of FGF-CX variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of FGF-CX variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential FGF-CX sequences is expressible as individual polypeptides, oralternatively, as a set of larger fusion proteins (e.g., for phagedisplay) containing the set of FGF-CX sequences therein. There are avariety of methods which can be used to produce libraries of potentialFGF-CX variants from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be performed in an automaticDNA synthesizer, and the synthetic gene then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision, in one mixture, of all of the sequences encoding the desiredset of potential FGF-CX sequences. Methods for synthesizing degenerateoligonucleotides are known in the art (see, e.g., Narang (1983)Tetrahedron 39:3; Itakura et al. (1984) Annu Rev Biochem 53:323;Itakuraet al. (1984) Science 198:1056; Ike et al. (1983) Nucl Acid Res11:477.

Polypeptide Libraries

In addition, libraries of fragments of the FGF-CX protein codingsequence can be used to generate a variegated population of FGF-CXfragments for screening and subsequent selection of variants of a FGF-CXprotein. In one embodiment, a library of coding sequence fragments canbe generated by treating a double stranded PCR fragment of a FGF-CXcoding sequence with a nuclease under conditions wherein nicking occursonly about once per molecule, denaturing the double stranded DNA,renaturing the DNA to form double stranded DNA that can includesense/antisense pairs from different nicked products, removing singlestranded portions from reformed duplexes by treatment with S1 nuclease,and ligating the resulting fragment library into an expression vector.By this method, an expression library can be derived which encodesN-terminal and internal fragments of various sizes of the FGF-CXprotein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries, for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of FGF-CX proteins. The mostwidely used techniques, which are amenable to high throughput analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recrusive ensemble mutagenesis (REM), a newtechnique that enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify FGF-CX variants (Arkin and Yourvan (1992) PNAS 89:7811-7815;Delgrave et al. (1993) Protein Engineering 6:327-331).

Anti-FGF-CX Antibodies

The invention further encompasses antibodies and antibody fragments,such as F_(ab) or (F_(ab))₂, that bind immunospecifically to any of theproteins of the invention.

An isolated FGF-CX protein, or a portion or fragment thereof, can beused as an immunogen to generate antibodies that bind FGF-CX usingstandard techniques for polyclonal and monoclonal antibody preparation.Full-length FGF-CX protein can be used. Alternatively, the inventionprovides antigenic peptide fragments of FGF-CX for use as immunogens.The antigenic peptide of FGF-CX comprises at least 4 amino acid residuesof the amino acid sequence shown in SEQ ID NO:2. The antigenic peptideencompasses an epitope of FGF-CX such that an antibody raised againstthe peptide forms a specific immune complex with FGF-CX. The antigenicpeptide may comprise at least 6 aa residues, at least 8 aa residues, atleast 10 aa residues, at least 15 aa residues, at least 20 aa residues,or at least 30 aa residues. In one embodiment of the invention, theantigenic peptide comprises a polypeptide comprising at least 6contiguous amino acids of SEQ ID NO:2.

In an embodiment of the invention, epitopes encompassed by the antigenicpeptide are regions of FGF-CX that are located on the surface of theprotein, e.g., hydrophilic regions. A hydrophobicity analysis of thehuman FGF-CX protein sequence, shown in FIG. 4, indicates that theregions between amino acids amino acids 30-50 and amino acids 120-195are particularly hydrophilic and, therefore, are likely to encodesurface residues useful for targeting antibody production. As a meansfor targeting antibody production, hydropathy plots showing regions ofhydrophilicity and hydrophobicity may be generated by any method wellknown in the art, including, for example, the Kyte Doolittle or the HoppWoods methods, either with or without Fourier transformation. See, e.g.,Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte andDoolittle 1982, J. Mol. Biol. 157: 105-142, each incorporated herein byreference in their entirety.

As disclosed herein, FGF-CX protein sequence of SEQ ID NO:2, orderivatives, fragments, analogs or homologs thereof, may be utilized asimmunogens in the generation of antibodies that immunospecifically-bindthese protein components. The term “antibody” as used herein refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site that specifically binds (immunoreacts with) an antigen,such as FGF-CX. Such antibodies include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, F_(ab) and F_((ab′)2)fragments, and an F_(ab) expression library. In a specific embodiment,antibodies to human FGF-CX proteins are disclosed. Various proceduresknown within the art may be used for the production of polyclonal ormonoclonal antibodies to a FGF-CX protein sequence of SEQ ID NO:2 orderivative, fragment, analog or homolog thereof. Some of these proteinsare discussed below.

For the production of polyclonal antibodies, various suitable hostanimals (e.g., rabbit, goat, mouse or other mammal) may be immunized byinjection with the native protein, or a synthetic variant thereof, or aderivative of the foregoing. An appropriate immunogenic preparation cancontain, for example, recombinantly expressed FGF-CX protein or achemically synthesized FGF-CX polypeptide. The preparation can furtherinclude an adjuvant. Various adjuvants used to increase theimmunological response include, but are not limited to, Freund's(complete and incomplete), mineral gels (e.g., aluminum hydroxide),surface active substances (e.g., lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, dinitrophenol, etc.), humanadjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum, orsimilar immunostimulatory agents. If desired, the antibody moleculesdirected against FGF-CX can be isolated from the mammal (e.g., from theblood) and further purified by well known techniques, such as protein Achromatography to obtain the IgG fraction.

The term “monoclonal antibody” or “monoclonal antibody composition”, asused herein, refers to a population of antibody molecules that containonly one species of an antigen binding site capable of immunoreactingwith a particular epitope of FGF-CX. A monoclonal antibody compositionthus typically displays a single binding affinity for a particularFGF-CX protein with which it immunoreacts. For preparation of monoclonalantibodies directed towards a particular FGF-CX protein, or derivatives,fragments, analogs or homologs thereof, any technique that provides forthe production of antibody molecules by continuous cell line culture maybe utilized. Such techniques include, but are not limited to, thehybridoma technique (see Kohler & Milstein, 1975 Nature 256: 495-497);the trioma technique; the human B-cell hybridoma technique (see Kozbor,et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique toproduce human monoclonal antibodies (see Cole, et al, 1985 In:MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Human monoclonal antibodies may be utilized in the practice ofthe present invention and may be produced by using human hybridomas (seeCote, et al, 1983. Proc Natl Acad Sci USA 80: 2026-2030) or bytransforming human B-cells with Epstein Barr Virus in vitro (see Cole,et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss,Inc., pp. 77-96). Each of the above citations are incorporated herein byreference in their entirety

According to the invention, techniques can be adapted for the productionof single-chain antibodies specific to a FGF-CX protein (see e.g., U.S.Pat. No. 4,946,778). In addition, methods can be adapted for theconstruction of F_(ab) expression libraries (see e.g., Huse, et al.,1989 Science 246: 1275-1281) to allow rapid and effective identificationof monoclonal F_(ab) fragments with the desired specificity for a FGF-CXprotein or derivatives, fragments, analogs or homologs thereof.Non-human antibodies can be “humanized” by techniques well known in theart. See e.g., U.S. Pat. No. 5,225,539. Each of the above citations areincorporated herein by reference. Antibody fragments that contain theidiotypes to a FGF-CX protein may be produced by techniques known in theart including, but not limited to: (i) an F_((ab′)2) fragment producedby pepsin digestion of an antibody molecule; (ii) an F_(ab) fragmentgenerated by reducing the disulfide bridges of an F_((ab′)2) fragment;(iii) an F_(ab) fragment generated by the treatment of the antibodymolecule with papain and a reducing agent and (iv) F_(v) fragments.

Additionally, recombinant anti-FGF-CX antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in PCTInternational Application No. PCT/US86/02269; European PatentApplication No. 184,187; European Patent Application No. 171,496;European Patent Application No. 173,494; PCT International PublicationNo. WO 86/01533; U.S. Pat. No. 4,816,567; European Patent ApplicationNo. 125,023; Better et al.(1988) Science 240:1041-1043; Liu et al.(1987) PNAS 84:3439-3443; Liu et al. (1987) J Immunol. 139:3521-3526;Sun et al. (1987) PNAS 84:214-218; Nishimura et al. (1987) Cancer Res47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw et al. (1988),J. Natl Cancer Inst 80:1553-1559); Morrison(1985) Science 229:1202-1207;Oi et al. (1986) BioTechniques 4:214; U.S. Pat. No. 5,225,539; Jones etal. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534;and Beidler et al. (1988) J Immunol 141:4053-4060. Each of the abovecitations are incorporated herein by reference.

In one embodiment, methods for the screening of antibodies that possessthe desired specificity include, but are not limited to, enzyme-linkedimmunosorbent assay (ELISA) and other immunologically-mediatedtechniques known within the art. In a specific embodiment, selection ofantibodies that are specific to a particular domain of a FGF-CX proteinis facilitated by generation of hybridomas that bind to the fragment ofa FGF-CX protein possessing such a domain. Antibodies that are specificfor one or more domains within a FGF-CX protein, e.g., the domainspanning the first fifty amino-terminal residues specific to FGF-CX whencompared to FGF-9, or derivatives, fragments, analogs or homologsthereof, are also provided herein.

Anti-FGF-CX antibodies may be used in methods known within the artrelating to the localization and/or quantitation of a FGF-CX protein(e.g. for use in measuring levels of the FGF-CX protein withinappropriate physiological samples, for use in diagnostic methods, foruse in imaging the protein, and the like). In a given embodiment,antibodies for FGF-CX proteins, or derivatives, fragments, analogs orhomologs thereof, that contain the antibody derived binding domain, areutilized as pharmacologically-active compounds [hereinafter“Therapeutics”].

An anti-FGF-CX antibody (e.g., monoclonal antibody) can be used toisolate FGF-CX by standard techniques, such as affinity chromatographyor immunoprecipitation. An anti-FGF-CX antibody can facilitate thepurification of natural FGF-CX from cells and of recombinantly producedFGF-CX expressed in host cells. Moreover, an anti-FGF-CX antibody can beused to detect FGF-CX protein (e.g., in a cellular lysate or cellsupernatant) in order to evaluate the abundance and pattern ofexpression of the FGF-CX protein. Anti-FGF-CX antibodies can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g. to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling (ie.,physically linking) the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

FGF-CX Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding FGF-CX protein,or derivatives, fragments, analogs or homologs thereof. As used herein,the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments can be ligated. Another type ofvector is a viral vector, wherein additional DNA segments can be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)are integrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “expression vectors”. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” can be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, that is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerthat allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to includes promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; GENE EXPRESSION TECHNOLOGY: METHODSIN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those that direct constitutive expression of anucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g. FGF-CX proteins, mutant forms ofFGF-CX, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of FGF-CX in prokaryotic or eukaryotic cells. For example,FGF-CX can be expressed in bacterial cells such as E. coli, insect cells(using baculovirus expression vectors) yeast cells or mammalian cells.Suitable host cells are discussed further in Goeddel, GENE EXPRESSIONTECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.(1990). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: (1) to increase expression ofrecombinant protein; (2) to increase the solubility of the recombinantprotein; and (3) to aid in the purification of the recombinant proteinby acting as a ligand in affmity purification. Often, in fusionexpression vectors, a proteolytic cleavage site is introduced at thejunction of the fusion moiety and the recombinant protein to enableseparation of the recombinant protein from the fusion moiety subsequentto purification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

Examples of suitable inducible non-fuision E. coli expression vectorsinclude pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif. (1990) 60-89).

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein. See, Gottesman, GENEEXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, SanDiego, Calif. (1990) 119-128. Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the FGF-CX expression vector is a yeastexpression vector. Examples of vectors for expression in yeast S.cerivisae include pYepSec1 (Baldari, et al., (1987) EMBO J 6:229-234),pMFa (Kuran and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz etal., (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego,Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).

Alternatively, FGF-CX can be expressed in insect cells using baculovirusexpression vectors. Baculovirus vectors available for expression ofproteins in cultured insect cells (e.g., SF9 cells) include the pAcseries (Smith et al. (1983) Mol Cell Biol 3:2156-2165) and the pVLseries (Lucklow and Summers (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840)and pMT2PC (Kaufmnan et al. (1987) EMBO J 6: 187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells. See, e.g., Chapters 16 and 17 ofSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv Immunol 43:235-275), in particular promoters of T cellreceptors (Winoto and Baltimore (1989) EMBO J 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, e.g., themurine hox promoters (Kessel and Gruss (1990) Science 249:374-379) andthe a-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to FGF-CX mRNA. Regulatory sequences operativelylinked to a nucleic acid cloned in the antisense orientation can bechosen that direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen that directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub etal., “Antisense RNA as a molecular tool for genetic analysis,”Reviews—Trends in Genetics, Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example,FGF-CX protein can be expressed in bacterial cells such as E. coli,insect cells, yeast or mammalian cells (such as Chinese hamster ovarycells (CHO) or COS cells). Other suitable host cells are known to thoseskilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MOLECULARCLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest. Variousselectable markers include those that confer resistance to drugs, suchas G418, hygromycin and methotrexate. Nucleic acid encoding a selectablemarker can be introduced into a host cell on the same vector as thatencoding FGF-CX or can be introduced on a separate vector. Cells stablytransfected with the introduced nucleic acid can be identified by drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) FGF-CX protein.Accordingly, the invention further provides methods for producing FGF-CXprotein using the host cells of the invention. In one embodiment, themethod comprises culturing the host cell of invention (into which arecombinant expression vector encoding FGF-CX has been introduced) in asuitable medium such that FGF-CX protein is produced. In anotherembodiment, the method further comprises isolating FGF-CX from themedium or the host cell.

Transgenic Animals

The host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichFGF-CX-coding sequences have been introduced. Such host cells can thenbe used to create non-human transgenic animals in which exogenous FGF-CXsequences have been introduced into their genome or homologousrecombinant animals in which endogenous FGF-CX sequences have beenaltered. Such animals are useful for studying the function and/oractivity of FGF-CX and for identifying and/or evaluating modulators ofFGF-CX activity. As used herein, a “transgenic animal” is a non-humananimal, preferably a mammal, more preferably a rodent such as a rat ormouse, in which one or more of the cells of the animal includes atransgene. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, amphibians, etc. Atransgene is exogenous DNA that is integrated into the genome of a cellfrom which a transgenic animal develops and that remains in the genomeof the mature animal, thereby directing the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal. As used herein, a “homologous recombinant animal” is a non-humananimal, preferably a mammal, more preferably a mouse, in which anendogenous FGF-CX gene has been altered by homologous recombinationbetween the endogenous gene and an exogenous DNA molecule introducedinto a cell of the animal, e.g., an embryonic cell of the animal, priorto development of the animal.

A transgenic animal of the invention can be created by introducingFGF-CX-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, retroviral infection, and allowing theoocyte to develop in a pseudopregnant female foster animal. The humanFGF-CX DNA sequence of SEQ ID NO:1 can be introduced as a transgene intothe genome of a non-human animal. Alternatively, a nonhuman homologue ofthe human FGF-CX gene, such as a mouse FGF-CX gene, can be isolatedbased on hybridization to the human FGF-CX cDNA (described furtherabove) and used as a transgene. Intronic sequences and polyadenylationsignals can also be included in the transgene to increase the efficiencyof expression of the transgene. A tissue-specific regulatory sequence(s)can be operably linked to the FGF-CX transgene to direct expression ofFGF-CX protein to particular cells. Methods for generating transgenicanimals via embryo manipulation and microinjection, particularly animalssuch as mice, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866; 4,870,009; and 4,873,191; andHogan 1986, In: MANIPULATING THE MOUSE EMBRYO, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. Similar methods are used forproduction of other transgenic animals. A transgenic founder animal canbe identified based upon the presence of the FGF-CX transgene in itsgenome and/or expression of FGF-CX mRNA in tissues or cells of theanimals. A transgenic founder animal can then be used to breedadditional animals carrying the transgene. Moreover, transgenic animalscarrying a transgene encoding FGF-CX can further be bred to othertransgenic animals carrying other transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a FGF-CX gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the FGF-CX gene. The FGF-CX gene can be a humangene (e.g., SEQ ID NO:1), but more preferably, is a non-human homologueof a human FGF-CX gene. For example, a mouse homologue of human FGF-CXgene of SEQ ID NO:1 can be used to construct a homologous recombinationvector suitable for altering an endogenous FGF-CX gene in the mousegenome. In one embodiment, the vector is designed such that, uponhomologous recombination, the endogenous FGF-CX gene is functionallydisrupted (i.e., no longer encodes a functional protein; also referredto as a “knock out” vector).

Alternatively, the vector can be designed such that, upon homologousrecombination, the endogenous FGF-CX gene is mutated or otherwisealtered but still encodes functional protein (e.g., the upstreamregulatory region can be altered to thereby alter the expression of theendogenous FGF-CX protein). In the homologous recombination vector, thealtered portion of the FGF-CX gene is flanked at its 5′ and 3′ ends byadditional nucleic acid of the FGF-CX gene to allow for homologousrecombination to occur between the exogenous FGF-CX gene carried by thevector and an endogenous FGF-CX gene in an embryonic stem cell. Theadditional flanking FGF-CX nucleic acid is of sufficient length forsuccessful homologous recombination with the endogenous gene. Typically,several kilobases of flanking DNA (both at the 5′ and 3′ ends) areincluded in the vector. See e.g., Thomas et al. (1987) Cell 51:503 for adescription of homologous recombination vectors. The vector isintroduced into an embryonic stem cell line (e.g. by electroporation)and cells in which the introduced FGF-CX gene has homologouslyrecombined with the endogenous FGF-CX gene are selected (see e.g., Li etal. (1992) Cell 69:915).

The selected cells are then injected into a blastocyst of an animal(e.g., a mouse) to form aggregation chimeras. See e.g., Bradley 1987,In: TERATOCARCINOMAS AND EMBRYONIC STEM CELLS: A PRACTICAL APPROACH,Robertson, ed. IRL, Oxford, pp. 113-152. A chimeric embryo can then beimplanted into a suitable pseudopregnant female foster animal and theembryo brought to term. Progeny harboring the homologously recombinedDNA in their germ cells can be used to breed animals in which all cellsof the animal contain the homologously recombined DNA by germlinetransnission of the transgene. Methods for constructing homologousrecombination vectors and homologous recombinant animals are describedfurther in Bradley (1991) Curr Opin Biotechnol 2:823-829; PCTInternational Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968;and WO 93/04169.

In another embodiment, transgenic non-humans animals can be producedthat contain selected systems that allow for regulated expression of thetransgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) PNAS 89:6232-6236.Another example of a recombinase system is the FLP recombinase system ofSaccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355.If a cre/loxP recombinase system is used to regulate expression of thetransgene, animals containing transgenes encoding both the Crerecombinase and a selected protein are required. Such animals can beprovided through the construction of “double” transgenic animals, e.g.,by mating two transgenic animals, one containing a transgene encoding aselected protein and the other containing a transgene encoding arecombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut et al. (1997)Nature 385:810-813. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G₀ phase. The quiescent cell can then be fused, e.g., throughthe use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated. Thereconstructed oocyte is then cultured such that it develops to morula orblastocyte and then transferred to pseudopregnant female foster animal.The offspring borne of this female foster animal will be a clone of theanimal from which the cell, e.g., the somatic cell, is isolated.

Pharmaceutical Compositions

The FGF-CX nucleic acid molecules, FGF-CX proteins, and anti-FGF-CXantibodies (also referred to herein as “active compounds”) of theinvention, and derivatives, fragments, analogs and homologs thereof, canbe incorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the nucleic acidmolecule, protein, or antibody and a pharmaceutically acceptablecarrier. As used herein, “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Suitable carriers are described in the most recent edition ofRemington's Pharmaceutical Sciences, a standard reference text in thefield, which is incorporated herein by reference. Preferred examples ofsuch carriers or diluents include, but are not limited to, water,saline, finger's solutions, dextrose solution, and 5% human serumalbumin. Liposomes and non-aqueous vehicles such as fixed oils may alsobe used. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe compositions is contemplated. Supplementary active compounds canalso be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetaaacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a FGF-CX protein or anti-FGF-CX antibody) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by any of a number of routes, e.g., as described in U.S.Pat. No. 5,703,055. Delivery can thus also include, e.g., intravenousinjection, local administration (see U.S. Pat. No. 5,328,470) orstereotactic injection (see e.g., Chen et al. (1994) PNAS 91:3054-3057).The pharmaceutical preparation of the gene therapy vector can includethe gene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells that producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods:(a) screening assays; (b) detection assays (e.g., chromosomal mapping,tissue typing, forensic biology), (c) predictive medicine (e.g.,diagnostic assays, prognostic assays, monitoring clinical trials, andpharmacogenomics); and (d) methods of treatment (e.g., therapeutic andprophylactic). As described herein, in one embodiment, a FGF-CX proteinof the invention has the ability to bind ATP.

The isolated nucleic acid molecules of the invention can be used toexpress FGF-CX protein (e.g., via a recombinant expression vector in ahost cell in gene therapy applications), to detect FGF-CX mRNA (e.g., ina biological sample) or a genetic lesion in a FGF-CX gene, and tomodulate FGF-CX activity, as described further below. In addition, theFGF-CX proteins can be used to screen drugs or compounds that modulatethe FGF-CX activity or expression as well as to treat disorderscharacterized by insufficient or excessive production of FGF-CX protein,for example proliferative or differentiative disorders, or production ofFGF-CX protein forms that have decreased or aberrant activity comparedto FGF-CX wild type protein. In addition, the anti-FGF-CX antibodies ofthe invention can be used to detect and isolate FGF-CX proteins andmodulate FGF-CX activity.

This invention further pertains to novel agents identified by the abovedescribed screening assays and uses thereof for treatments as describedherein.

Screening Assays

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)that bind to FGF-CX proteins or have a stimulatory or inhibitory effecton, for example, FGF-CX expression or FGF-CX activity.

In one embodiment, the invention provides assays for screening candidateor test compounds which bind to or modulate the activity of a FGF-CXprotein or polypeptide or biologically active portion thereof. The testcompounds of the present invention can be obtained using any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the “one-bead one-compound” library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam (1997) Anticancer Drug Des 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc Natl AcadSci U.S.A. 90:6909; Erb et al. (1994) Proc Natl Acad Sci U.S.A.91:11422; Zuckermann et al. (1994) J Med Chem 37:2678; Cho et al. (1993)Science 261:1303; Carrell et al. (1994) Angew Chem Int Ed Engl 33:2059;Carell et al. (1994) Angew Chem Int Ed Engl 33:2061; and Gallop et al.(1994) J Med Chem 37:1233.

Libraries of compounds may be presented in solution (e.g. Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), on chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner USP '409), plasmids (Cull etal. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott andSmith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406;Cwirla et al. (1990) Proc Natl Acad Sci U.S.A. 87:6378-6382; Felici(1991) J Mol Biol 222:301-310; Ladner above.).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a membrane-bound form of FGF-CX protein, or a biologicallyactive portion thereof, on the cell surface is contacted with a testcompound and the ability of the test compound to bind to a FGF-CXprotein determined. The cell, for example, can of mammalian origin or ayeast cell. Determining the ability of the test compound to bind to theFGF-CX protein can be accomplished, for example, by coupling the testcompound with a radioisotope or enzymatic label such that binding of thetest compound to the FGF-CX protein or biologically active portionthereof can be determined by detecting the labeled compound in acomplex. For example, test compounds can be labeled with ¹²⁵I, ³⁵S, ¹⁴C,or ³H, either directly or indirectly, and the radioisotope detected bydirect counting of radioemission or by scintillation counting.Alternatively, test compounds can be enzymatically labeled with, forexample, horseradish peroxidase, alkaline phosphatase, or luciferase,and the enzymatic label detected by determination of conversion of anappropriate substrate to product. In one embodiment, the assay comprisescontacting a cell which expresses a membrane-bound form of FGF-CXprotein, or a biologically active portion thereof, on the cell surfacewith a known compound which binds FGF-CX to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with a FGF-CX protein, whereindetermining the ability of the test compound to interact with a FGF-CXprotein comprises determining the ability of the test compound topreferentially bind to FGF-CX or a biologically active portion thereofas compared to the known compound.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a membrane-bound form of FGF-CX protein, ora biologically active portion thereof, on the cell surface with a testcompound and determining the ability of the test compound to modulate(e.g., stimulate or inhibit) the activity of the FGF-CX protein orbiologically active portion thereof. Determining the ability of the testcompound to modulate the activity of FGF-CX or a biologically activeportion thereof can be accomplished, for example, by determining theability of the FGF-CX protein to bind to or interact with a FGF-CXtarget molecule. As used herein, a “target molecule” is a molecule withwhich a FGF-CX protein binds or interacts in nature, for example, amolecule on the surface of a cell which expresses a FGF-CX interactingprotein, a molecule on the surface of a second cell, a molecule in theextracellular milieu, a molecule associated with the internal surface ofa cell membrane or a cytoplasmic molecule. A FGF-CX target molecule canbe a non-FGF-CX molecule or a FGF-CX protein or polypeptide of thepresent invention. In one embodiment, a FGF-CX target molecule is acomponent of a signal transduction pathway that facilitates transductionof an extracellular signal (e.g., a signal generated by binding of acompound to a membrane-bound FGF-CX molecule) through the cell membraneand into the cell. The target, for example, can be a secondintercellular protein that has catalytic activity or a protein thatfacilitates the association of downstream signaling molecules withFGF-CX.

Determining the ability of the FGF-CX protein to bind to or interactwith a FGF-CX target molecule can be accomplished by one of the methodsdescribed above for determining direct binding. In one embodiment,determining the ability of the FGF-CX protein to bind to or interactwith a FGF-CX target molecule can be accomplished by determining theactivity of the target molecule. For example, the activity of the targetmolecule can be determined by detecting induction of a cellular secondmessenger of the target (ie. intracellular Ca²⁺, diacylglycerol, IP₃,etc.), detecting catalytic/enzymatic activity of the target anappropriate substrate, detecting the induction of a reporter gene(comprising a FGF-CX-responsive regulatory element operatively linked toa nucleic acid encoding a detectable marker, e.g., luciferase), ordetecting a cellular response, for example, cell survival, cellulardifferentiation, or cell proliferation.

In yet another embodiment, an assay of the present invention is acell-free assay comprising contacting a FGF-CX protein or biologicallyactive portion thereof with a test compound and determining the abilityof the test compound to bind to the FGF-CX protein or biologicallyactive portion thereof. Binding of the test compound to the FGF-CXprotein can be determined either directly or indirectly as describedabove. In one embodiment, the assay comprises contacting the FGF-CXprotein or biologically active portion thereof with a known compoundwhich binds FGF-CX to form an assay mixture, contacting the assaymixture with a test compound, and determining the ability of the testcompound to interact with a FGF-CX protein, wherein determining theability of the test compound to interact with a FGF-CX protein comprisesdetermining the ability of the test compound to preferentially bind toFGF-CX or biologically active portion thereof as compared to the knowncompound.

In another embodiment, an assay is a cell-free assay comprisingcontacting FGF-CX protein or biologically active portion thereof with atest compound and determining the ability of the test compound tomodulate (e.g., stimulate or inhibit) the activity of the FGF-CX proteinor biologically active portion thereof. Determining the ability of thetest compound to modulate the activity of FGF-CX can be accomplished,for example, by determining the ability of the FGF-CX protein to bind toa FGF-CX target molecule by one of the methods described above fordetermining direct binding. In an alternative embodiment, determiningthe ability of the test compound to modulate the activity of FGF-CX canbe accomplished by determining the ability of the FGF-CX protein furthermodulate a FGF-CX target molecule. For example, the catalytic/enzymaticactivity of the target molecule on an appropriate substrate can bedetermined as previously described.

In yet another embodiment, the cell-free assay comprises contacting theFGF-CX protein or biologically active portion thereof with a knowncompound which binds FGF-CX to form an assay mixture, contacting theassay mixture with a test compound, and determining the ability of thetest compound to interact with a FGF-CX protein, wherein determining theability of the test compound to interact with a FGF-CX protein comprisesdetermining the ability of the FGF-CX protein to preferentially bind toor modulate the activity of a FGF-CX target molecule.

The cell-free assays of the present invention are amenable to use ofboth the soluble form or the membrane-bound form of FGF-CX. In the caseof cell-free assays comprising the membrane-bound form of FGF-CX, it maybe desirable to utilize a solubilizing agent such that themembrane-bound form of FGF-CX is maintained in solution. Examples ofsuch solubilizing agents include non-ionic detergents such asn-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_(n),N-dodecyl-N,N-dimethyl-3-ammonio-1 -propane sulfonate,3-(3-cholamidopropyl)dimethylamminiol-1-propane sulfonate (CHAPS), or3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate(CHAPSO).

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either FGF-CX or its targetmolecule to facilitate separation of complexed from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay. Binding of a test compound to FGF-CX, or interaction of FGF-CXwith a target molecule in the presence and absence of a candidatecompound, can be accomplished in any vessel suitable for containing thereactants. Examples of such vessels include microtiter plates, testtubes, and micro-centrifuge tubes. In one embodiment, a fusion proteincan be provided that adds a domain that allows one or both of theproteins to be bound to a matrix. For example, GST-FGF-CX fusionproteins or GST-target fusion proteins can be adsorbed onto glutathionesepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathionederivatized microtiter plates, that are then combined with the testcompound or the test compound and either the non-adsorbed target proteinor FGF-CX protein, and the mixture is incubated under conditionsconducive to complex formation (e.g., at physiological conditions forsalt and pH). Following incubation, the beads or microtiter plate wellsare washed to remove any unbound components, the matrix immobilized inthe case of beads, complex determined either directly or indirectly, forexample, as described above. Alternatively, the complexes can bedissociated from the matrix, and the level of FGF-CX binding or activitydetermined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either FGF-CX orits target molecule can be immobilized utilizing conjugation of biotinand streptavidin. Biotinylated FGF-CX or target molecules can beprepared from biotin-NHS (N-hydroxy-succinimide) using techniques wellknown in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,Ill.), and immobilized in the wells of streptavidin-coated 96 wellplates (Pierce Chemical). Alternatively, antibodies reactive with FGF-CXor target molecules, but which do not interfere with binding of theFGF-CX protein to its target molecule, can be derivatized to the wellsof the plate, and unbound target or FGF-CX trapped in the wells byantibody conjugation. Methods for detecting such complexes, in additionto those described above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with the FGF-CXor target molecule, as well as enzyme-linked assays that rely ondetecting an enzymatic activity associated with the FGF-CX or targetmolecule.

In another embodiment, modulators of FGF-CX expression are identified ina method wherein a cell is contacted with a candidate compound and theexpression of FGF-CX mRNA or protein in the cell is determined. Thelevel of expression of FGF-CX mRNA or protein in the presence of thecandidate compound is compared to the level of expression of FGF-CX mRNAor protein in the absence of the candidate compound. The candidatecompound can then be identified as a modulator of FGF-CX expressionbased on this comparison. For example, when expression of FGF-CX mRNA orprotein is greater (statistically significantly greater) in the presenceof the candidate compound than in its absence, the candidate compound isidentified as a stimulator of FGF-CX mRNA or protein expression.Alternatively, when expression of FGF-CX mRNA or protein is less(statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of FGF-CX mRNA or protein expression. The level of FGF-CX mRNAor protein expression in the cells can be determined by methodsdescribed herein for detecting FGF-CX mRNA or protein.

In yet another aspect of the invention, the FGF-CX proteins can be usedas “bait proteins” in a two-hybrid assay or three hybrid assay (see,e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232;Madura et al. (1993) J Biol Chem 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696;and Brent WO94/10300), to identify other proteins that bind to orinteract with FGF-CX (“FGF-CX-binding proteins” or “FGF-CX-bp”) andmodulate FGF-CX activity. Such FGF-CX-binding proteins are also likelyto be involved in the propagation of signals by the FGF-CX proteins as,for example, upstream or downstream elements of the FGF-CX pathway.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for FGF-CX is fused toa gene encoding the DNA binding domain of a known transcription factor(e.g., GAL-4). In the other construct, a DNA sequence, from a library ofDNA sequences, that encodes an unidentified protein (“prey” or “sample”)is fused to a gene that codes for the activation domain of the knowntranscription factor. If the “bait” and the “prey” proteins are able tointeract, in vivo, forming a FGF-CX-dependent complex, the DNA-bindingand activation domains of the transcription factor are brought intoclose proximity. This proximity allows transcription of a reporter gene(e.g., LacZ) that is operably linked to a transcriptional regulatorysite responsive to the transcription factor. Expression of the reportergene can be detected and cell colonies containing the functionaltranscription factor can be isolated and used to obtain the cloned genethat encodes the protein which interacts with FGF-CX.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(i) map their respective genes on a chromosome; and, thus, locate generegions associated with genetic disease; (ii) identify an individualfrom a minute biological sample (tissue typing); and (iii) aid inforensic identification of a biological sample.

The FGF-CX sequences of the present invention can also be used toidentify individuals from minute biological samples. In this technique,an individual's genomic DNA is digested with one or more restrictionenzymes, and probed on a Southern blot to yield unique bands foridentification. The sequences of the present invention are useful asadditional DNA markers for RFLP (“restriction fragment lengthpolymorphisms,” described in U.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique that determines the actual base-by-baseDNA sequence of selected portions of an individual's genome. Thus, theFGF-CX sequences described herein can be used to prepare two PCR primersfrom the 5′ and 3′ ends of the sequences. These primers can then be usedto amplify an individual's DNA and subsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The FGF-CX sequences of the invention uniquely represent portions of thehuman genome. Allelic variation occurs to some degree in the codingregions of these sequences, and to a greater degree in the noncodingregions. It is estimated that allelic variation between individualhumans occurs with a frequency of about once per each 500 bases. Much ofthe allelic variation is due to single nucleotide polymorphisms (SNPs),which include restriction fragment length polymorphisms (RFLPs).

Each of the sequences described herein can, to some degree, be used as astandard against which DNA from an individual can be compared foridentification purposes. Because greater numbers of polymorphisms occurin the noncoding regions, fewer sequences are necessary to differentiateindividuals. The noncoding sequences of SEQ ID NO:1, as described above,can comfortably provide positive individual identification with a panelof perhaps 10 to 1,000 primers that each yield a noncoding amplifiedsequence of 100 bases. If predicted coding sequences are used, a moreappropriate number of primers for positive individual identificationwould be 500-2,000.

Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, pharmacogenomics, andmonitoring clinical trials are used for prognostic (predictive) purposesto thereby treat an individual prophylactically. Accordingly, one aspectof the present invention relates to diagnostic assays for determiningFGF-CX protein and/or nucleic acid expression as well as FGF-CXactivity, in the context of a biological sample (e.g., blood, serum,cells, tissue) to thereby determine whether an individual is afflictedwith a disease or disorder, or is at risk of developing a disorder,associated with aberrant FGF-CX expression or activity. The inventionalso provides for prognostic (or predictive) assays for determiningwhether an individual is at risk of developing a disorder associatedwith FGF-CX protein, nucleic acid expression or activity. For example,mutations in a FGF-CX gene can be assayed in a biological sample. Suchassays can be used for prognostic or predictive purpose to therebyprophylactically treat an individual prior to the onset of a disordercharacterized by or associated with FGF-CX protein, nucleic acidexpression or activity.

Another aspect of the invention provides methods for determining FGF-CXprotein, nucleic acid expression or FGF-CX activity in an individual tothereby select appropriate therapeutic or prophylactic agents for thatindividual (referred to herein as “pharmacogenomics”). Pharmacogenomicsallows for the selection of agents (e.g., drugs) for therapeutic orprophylactic treatment of an individual based on the genotype of theindividual (e.g., the genotype of the individual examined to determinethe ability of the individual to respond to a particular agent.)

Yet another aspect of the invention pertains to monitoring the influenceof agents (e.g., drugs, compounds) on the expression or activity ofFGF-CX in clinical trials.

These and other agents are described in further detail in the followingsections.

Diagnostic Assays

Fibroblast growth factors FGF-1 through FGF-9 generally promote cellproliferation in cells carrying the particular growth factor receptor.Examples of FGF growth promotion include epithelial cells, such asfibroblasts and keratinocytes, in the anterior eye after surgery. Otherconditions in which proliferation of cells plays a role include tumors,restenosis, psoriasis, Dupuytren's contracture, diabetic complications,Kaposi's sarcoma and rheumatoid arthritis.

FGF-CX may be used in the method of the invention for detecting itscorresponding fibroblast growth factor receptor CX (FGFRCX) in a sampleor tissue. The method comprises contacting the sample or tissue withFGF-CX, allowing formation of receptor-ligand pairs, and detecting anyFGFRCX: FGF-CX pairs. Compositions containing FGF-CX can be used toincrease FGFRCX activity, for example to stimulate cartilage or bonerepair. Compositions containing FGF-CX antagonists or FGF-CX bindingagents (e.g. anti- FGF-CX antibodies) can be used to treat diseasescaused by an excess of FGF-CX or overactivity of FGFRCX, especiallymultiple or solitary hereditary exostosis, hallux valgus deformity,achondroplasia, synovial chondromatosis and endochondromas.

Glia activating factor (GAF) and the DNA encoding GAF act tospecifically promote growth of glial cells. Some examples ofglia-associated disorders in which GAF may be utilized to modulate glialcell activities are cerebral lesions, cerebral edema, senile dementia,Alzheimer's disease, diabetic neuropathies, etc. Similarly, FGF-CX maybe used in diagnosis or treating glial cell related disorders. Theglial-cell modulating activity of FGF-CX may be as aneuroprotective-like activity, and FGF-CX may be used as aneuroprotective agent. Due to the close homology of FGF-CX to FGF-9,which was identified originally as a glia activating factor, it can bepresumed that the FGF-CX sequence is also a glia activating factor.FGF-CX can therefor be used to stimulate the growth of glia cells andcan be used to accelerate healing of cerebral lesions or to treatcerebral edema, senile dementia, Alzheimer's disease, or diabeticneuropathy.

FGF-CX can also be used to stimulates fibroblasts (for acceleratinghealing of burns, wounds, ulcers, etc), megakaryocytes (to increase thenumber of platelets), hematopoietic cells, immune system cells, andvascular smooth muscle cells. FGF-CX is also expected to haveosteogenesis-promoting activity, and can be used for treating bonefractures and osteoporosis. Assay of FGF-CX polypeptide or nucleic acidmoieties may be useful in diagnosis of cerebral tumors, and antibodiesagainst could be used to treat such tumors. It can also be used as areagent for stimulating growth of cultured cells. An anticipated dosageis 1 ng-0.1 mg/kg/day, though treatment may vary depending on the typeor severity of the disorder being treated. FGF-CX polypeptides may beused as platelet increasing agents, osteogenesis promoting agents or fortreating cerebral nervous diseases or hepatopathy such as hepaticcirrhosis. They can also be used to treat cancer when used alongside ananticancer agent. Antibodies directed against the FGF-CX polypeptide, orfragments, derivatives, or analogs thereof, can be used for detecting ordetermining a biological activity of a FGF-CX polypeptide or forpurifying a FGF-CX polypeptide. Those antibodies that also neutralizethe cell growth activity of FGF-CX can be used as anticancer agents.

Many, if not all, homologous proteins are known in the art to haveclosely related or identical functions. See, e.g., Lewin, “Chapter 21:Structural Genes Belong to Families” In: GENES II, 1985, John Wiley andSons, Inc., New York. The FGF-CX polypeptide closely resembles theXenopus XFGF-20 protein, which was shown previously to be specificallyexpressed in highly proliferative tissues (see, e.g., Koga et al.,above). Therefore, it is presumed that FGF-CX would also modulatecellular activity in highly proliferative tissues. FGF-CX may thus beparticularly useful in diagnosing proliferative disorders and instimulating the growth of cells and tissues in order to overcomepathological states in which such growth has been suppressed orinhibited. Oligonucleotides corresponding to any one portion of theFGF-CX nucleic acids of SEQ ID NO:1 may be used to detect the expressionof a FGF-CX-like gene. The proteins of the invention may be used tostimulate production of antibodies specifically binding the proteins.Such antibodies may be used in immunodiagnostic procedures to detect theoccurrence of the protein in a sample. The proteins of the invention maybe used to stimulate cell growth and cell proliferation in conditions inwhich such growth would be favorable. An example would be to counteracttoxic side effects of chemotherapeutic agents on, for example,hematopoiesis and platelet formation, linings of the gastrointestinaltract, and hair follicles. They may also be used to stimulate new cellgrowth in neurological disorders including, for example, Alzheimer'sdisease. Alternatively, antagonistic treatments may be administered inwhich an antibody specifically binding the FGF-CX-like proteins of theinvention would abrogate the specific growth-inducing effects of theproteins. Such antibodies may be useful, for example, in the treatmentof proliferative disorders including various tumors and benignhyperplasias.

An exemplary method for detecting the presence or absence of FGF-CX in abiological sample involves obtaining a biological sample from a testsubject and contacting the biological sample with a compound or an agentcapable of detecting FGF-CX protein or nucleic acid (e.g., mRNA, genomicDNA) that encodes FGF-CX protein such that the presence of FGF-CX isdetected in the biological sample. An agent for detecting FGF-CX mRNA orgenomic DNA is a labeled nucleic acid probe capable of hybridizing toFGF-CX mRNA or genomic DNA. The nucleic acid probe can be, for example,a full-length FGF-CX nucleic acid, such as the nucleic acid of SEQ IDNO:1, or a portion thereof, such as an oligonucleotide of at least 15,30, 50, 100, 250 or 500 nucleotides in length and sufficient tospecifically hybridize under stringent conditions to FGF-CX mRNA orgenomic DNA, as described above. Other suitable probes for use in thediagnostic assays of the invention are described herein.

An agent for detecting FGF-CX protein is an antibody capable of bindingto FGF-CX protein, preferably an antibody with a detectable label.Antibodies can be polyclonal, or more preferably, monoclonal. An intactantibody, or a fragment thereof (e.g., Fab or F(ab′)₂) can be used. Theterm “labeled”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin. The term“biological sample” is intended to include tissues, cells and biologicalfluids isolated from a subject, as well as tissues, cells and fluidspresent within a subject. That is, the detection method of the inventioncan be used to detect FGF-CX mRNA, protein, or genomic DNA in abiological sample in vitro as well as in vivo. For example, in vitrotechniques for detection of FGF-CX mRNA include Northern hybridizationsand in situ hybridizations. In vitro techniques for detection of FGF-CXprotein include enzyme linked immunosorbent assays (ELISAs), Westernblots, immunoprecipitations and immunofluorescence. In vitro techniquesfor detection of FGF-CX genomic DNA include Southern hybridizations.Furthermore, in vivo techniques for detection of FGF-CX protein includeintroducing into a subject a labeled anti-FGF-CX antibody. For example,the antibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A preferred biological sample is a peripheral blood leukocytesample isolated by conventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting FGF-CX protein, mRNA, orgenomic DNA, such that the presence of FGF-CX protein, mRNA or genomicDNA is detected in the biological sample, and comparing the presence ofFGF-CX protein, mRNA or genomic DNA in the control sample with thepresence of FGF-CX protein, mRNA or genomic DNA in the test sample.

The invention also encompasses kits for detecting the presence of FGF-CXin a biological sample. For example, the kit can comprise: a labeledcompound or agent capable of detecting FGF-CX protein or mRNA in abiological sample; means for determining the amount of FGF-CX in thesample; and means for comparing the amount of FGF-CX in the sample witha standard. The compound or agent can be packaged in a suitablecontainer. The kit can further comprise instructions for using the kitto detect FGF-CX protein or nucleic acid.

Prognostic Assays

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant FGF-CX expression or activity. For example, theassays described herein, such as the preceding diagnostic assays or thefollowing assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with FGF-CX protein, nucleicacid expression or activity in, e.g. proliferative or differentiativedisorders such as hyperplasias, tumors, restenosis, psoriasis,Dupuytren's contracture, diabetic complications, or rheumatoidarthritis, etc.; and glia-associated disorders such as cerebral lesions,diabetic neuropathies, cerebral edema, senile dementia, Alzheimer'sdisease, etc. Alternatively, the prognostic assays can be utilized toidentify a subject having or at risk for developing a disease ordisorder. Thus, the present invention provides a method for identifyinga disease or disorder associated with aberrant FGF-CX expression oractivity in which a test sample is obtained from a subject and FGF-CXprotein or nucleic acid (e.g, mRNA, genomic DNA) is detected, whereinthe presence of FGF-CX protein or nucleic acid is diagnostic for asubject having or at risk of developing a disease or disorder associatedwith aberrant FGF-CX expression or activity. As used herein, a “testsample” refers to a biological sample obtained from a subject ofinterest. For example, a test sample can be a biological fluid (e.g.,serum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant FGF-CX expression or activity. For example,such methods can be used to determine whether a subject can beeffectively treated with an agent for a disorder, such as aproliferative disorder, differentiative disorder, glia-associateddisorders, etc. Thus, the present invention provides methods fordetermining whether a subject can be effectively treated with an agentfor a disorder associated with aberrant FGF-CX expression or activity inwhich a test sample is obtained and FGF-CX protein or nucleic acid isdetected (e.g. wherein the presence of FGF-CX protein or nucleic acid isdiagnostic for a subject that can be administered the agent to treat adisorder associated with aberrant FGF-CX expression or activity.)

The methods of the invention can also be used to detect genetic lesionsin a FGF-CX gene, thereby determining if a subject with the lesionedgene is at risk for, or suffers from, a proliferative disorder,differentiative disorder, glia-associated disorder, etc. In variousembodiments, the methods include detecting, in a sample of cells fromthe subject, the presence or absence of a genetic lesion characterizedby at least one of an alteration affecting the integrity of a geneencoding a FGF-CX-protein, or the mis-expression of the FGF-CX gene. Forexample, such genetic lesions can be detected by ascertaining theexistence of at least one of (1) a deletion of one or more nucleotidesfrom a FGF-CX gene; (2) an addition of one or more nucleotides to aFGF-CX gene; (3) a substitution of one or more nucleotides of a FGF-CXgene, (4) a chromosomal rearrangement of a FGF-CX gene; (5) analteration in the level of a messenger RNA transcript of a FGF-CX gene,(6) aberrant modification of a FGF-CX gene, such as of the methylationpattern of the genomic DNA, (7) the presence of a non-wild type splicingpattern of a messenger RNA transcript of a FGF-CX gene, (8) a non-wildtype level of a FGF-CX-protein, (9) allelic loss of a FGF-CX gene, and(10) inappropriate post-translational modification of a FGF-CX-protein.As described herein, there are a large number of assay techniques knownin the art which can be used for detecting lesions in a FGF-CX gene. Apreferred biological sample is a peripheral blood leukocyte sampleisolated by conventional means from a subject. However, any biologicalsample containing nucleated cells may be used, including, for example,buccal mucosal cells.

In certain embodiments, detection of the lesion involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) PNAS91:360-364), the latter of which can be particularly useful fordetecting point mutations in the FGF-CX-gene (see Abravaya et al. (1995)Nucl Acids Res 23:675-682). This method can include the steps ofcollecting a sample of cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers that specificallyhybridize to a FGF-CX gene under conditions such that hybridization andamplification of the FGF-CX gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample. It is anticipated that PCR and/or LCR may be desirable to use asa preliminary amplification step in conjunction with any of thetechniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli et al., 1990, Proc Natl Acad Sci USA87:1874-1878), transcriptional amplification system (Kwoh, et al., 1989,Proc Natl Acad Sci USA 86:1173-1177), Q-Beta Replicase (Lizardi et al,1988, BioTechnology 6:1197), or any other nucleic acid amplificationmethod, followed by the detection of the amplified molecules usingtechniques well known to those of skill in the art. These detectionschemes are especially useful for the detection of nucleic acidmolecules if such molecules are present in very low numbers.

In an alternative embodiment, mutations in a FGF-CX gene from a samplecell can be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, for example, U.S. Pat. No. 5,493,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in FGF-CX can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA, tohigh density arrays containing hundreds or thousands of oligonucleotidesprobes (Cronin et al. (1996) Human Mutation 7: 244-255; Kozal et al.(1996) Nature Medicine 2: 753-759). For example, genetic mutations inFGF-CX can be identified in two dimensional arrays containinglight-generated DNA probes as described in Cronin et al. above. Briefly,a first hybridization array of probes can be used to scan through longstretches of DNA in a sample and control to identify base changesbetween the sequences by making linear arrays of sequential overlappingprobes. This step allows the identification of point mutations. Thisstep is followed by a second hybridization array that allows thecharacterization of specific mutations by using smaller, specializedprobe arrays complementary to all variants or mutations detected. Eachmutation array is composed of parallel probe sets, one complementary tothe wild-type gene and the other complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the FGF-CX gene anddetect mutations by comparing the sequence of the sample FGF-CX with thecorresponding wild-type (control) sequence. Examples of sequencingreactions include those based on techniques developed by Maxim andGilbert (1977) PNAS 74:560 or Sanger (1977) PNAS 74:5463. It is alsocontemplated that any of a variety of automated sequencing procedurescan be utilized when performing the diagnostic assays (Naeve et al.,(1995) Biotechniques 19:448), including sequencing by mass spectrometry(see, e.g., PCT International Publ. No. WO 94/16101; Cohen et al. (1996)Adv Chromatogr 36:127-162; and Griffin et al. (1993) Appl BiochemBiotechnol 38:147-159).

Other methods for detecting mutations in the FGF-CX gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242). In general, the art technique of “mismatch cleavage” startsby providing heteroduplexes of formed by hybridizing (labeled) RNA orDNA containing the wild-type FGF-CX sequence with potentially mutant RNAor DNA obtained from a tissue sample. The double-stranded duplexes aretreated with an agent that cleaves single-stranded regions of the duplexsuch as which will exist due to basepair mismatches between the controland sample strands. For instance, RNA/DNA duplexes can be treated withRNase and DNA/DNA hybrids treated with S1 nuclease to enzymaticallydigesting the mismatched regions. In other embodiments, either DNA/DNAor RNA/DNA duplexes can be treated with hydroxylamine or osmiumtetroxide and with piperidine in order to digest mismatched regions.After digestion of the mismatched regions, the resulting material isthen separated by size on denaturing polyacrylamide gels to determinethe site of mutation. See, for example, Cotton et al (1988) Proc NatlAcad Sci USA 85:4397; Saleeba et al (1992) Methods Enzymol 217:286-295.In an embodiment, the control DNA or RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in FGF-CX cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on a FGF-CXsequence, e.g., a wild-type FGF-CX sequence, is hybridized to a cDNA orother DNA product from a test cell(s). The duplex is treated with a DNAmismatch repair enzyme, and the cleavage products, if any, can bedetected from electrophoresis protocols or the like. See, for example,U.S. Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in FGF-CX genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl Acad Sci USA: 86:2766, see also Cotton(1993) Mutat Res 285:125-144; Hayashi (1992) Genet Anal Tech Appl9:73-79). Single-stranded DNA fragments of sample and control FGF-CXnucleic acids will be denatured and allowed to renature. The secondarystructure of single-stranded nucleic acids varies according to sequence,the resulting alteration in electrophoretic mobility enables thedetection of even a single base change. The DNA fragments may be labeledor detected with labeled probes. The sensitivity of the assay may beenhanced by using RNA, rather than DNA, in which the secondary structureis more sensitive to a change in sequence. In one embodiment, thesubject method utilizes heteroduplex analysis to separate doublestranded heteroduplex molecules on the basis of changes inelectrophoretic mobility. See, e.g., Keen et al. (1991) Trends Genet7:5.

In yet another embodiment the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE). See, e.g., Myerset al (1985) Nature 313:495. When DGGE is used as the method ofanalysis, DNA will be modified to insure that it does not completelydenature, for example by adding a GC clamp of approximately 40 bp ofhigh-melting GC-rich DNA by PCR. In a further embodiment, a temperaturegradient is used in place of a denaturing gradient to identifydifferences in the mobility of control and sample DNA. See, e.g.,Rosenbaum and Reissner (1987) Biophys Chem 265:12753.

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditions thatpermit hybridization only if a perfect match is found. See, e.g., Saikiet al. (1986) Nature 324:163); Saiki et al. (1989) Proc Natl Acad. SciUSA 86:6230. Such allele specific oligonucleotides are hybridized to PCRamplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology that depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection. See,e.g., Gasparini et al (1992) Mol Cell Probes 6:1. It is anticipated thatin certain embodiments amplification may also be performed using Taqligase for amplification. See, e.g., Barany (1991) Proc Natl Acad SciUSA 88:189. In such cases, ligation will occur only if there is aperfect match at the 3′ end of the 5′ sequence, making it possible todetect the presence of a known mutation at a specific site by lookingfor the presence or absence of amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a FGF-CX gene.

Furthermore, any cell type or tissue, preferably peripheral bloodleukocytes, in which FGF-CX is expressed may be utilized in theprognostic assays described herein. However, any biological samplecontaining nucleated cells may be used, including, for example, buccalmucosal cells.

Pharmacogenomics

Agents, or modulators that have a stimulatory or inhibitory effect onFGF-CX activity (e.g., FGF-CX gene expression), as identified by ascreening assay described herein can be administered to individuals totreat (prophylactically or therapeutically) disorders (e.g.,neurological, cancer-related or gestational disorders) associated withaberrant FGF-CX activity. In conjunction with such treatment, thepharmacogenomics (i.e., the study of the relationship between anindividual's genotype and that individual's response to a foreigncompound or drug) of the individual may be considered. Differences inmetabolism of therapeutics can lead to severe toxicity or therapeuticfailure by altering the relation between dose and blood concentration ofthe pharmacologically active drug. Thus, the pharmacogenomics of theindividual permits the selection of effective agents (e.g., drugs) forprophylactic or therapeutic treatments based on a consideration of theindividual's genotype. Such pharmacogenomics can further be used todetermine appropriate dosages and therapeutic regimens. Accordingly, theactivity of FGF-CX protein, expression of FGF-CX nucleic acid, ormutation content of FGF-CX genes in an individual can be determined tothereby select appropriate agent(s) for therapeutic or prophylactictreatment of the individual.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See e.g., Eichelbaum, 1996, Clin ExpPharmacol Physiol, 23:983-985 and Linder, 1997, Clin Chem, 43:254-266.In general, two types of pharmacogenetic conditions can bedifferentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body (altered drug action) or geneticconditions transmitted as single factors altering the way the body actson drugs (altered drug metabolism). These pharmacogenetic conditions canoccur either as rare defects or as polymorphisms. For example,glucose-6-phosphate dehydrogenase (G6PD) deficiency is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Thus, the activity of FGF-CX protein, expression of FGF-CX nucleic acid,or mutation content of FGF-CX genes in an individual can be determinedto thereby select appropriate agent(s) for therapeutic or prophylactictreatment of the individual. In addition, pharmacogenetic studies can beused to apply genotyping of polymorphic alleles encodingdrug-metabolizing enzymes to the identification of an individual's drugresponsiveness phenotype. This knowledge, when applied to dosing or drugselection, can avoid adverse reactions or therapeutic failure and thusenhance therapeutic or prophylactic efficiency when treating a subjectwith a FGF-CX modulator, such as a modulator identified by one of theexemplary screening assays described herein.

Monitoring Clinical Efficacy

Monitoring the influence of agents (e.g., drugs, compounds) on theexpression or activity of FGF-CX (e.g., the ability to modulate aberrantcell proliferation and/or differentiation) can be applied in basic drugscreening and in clinical trials. For example, the effectiveness of anagent determined by a screening assay as described herein to increaseFGF-CX gene expression, protein levels, or upregulate FGF-CX activity,can be monitored in clinical trials of subjects exhibiting decreasedFGF-CX gene expression, protein levels, or downregulated FGF-CXactivity. Alternatively, the effectiveness of an agent determined by ascreening assay to decrease FGF-CX gene expression, protein levels, ordownregulate FGF-CX activity, can be monitored in clinical trials ofsubjects exhibiting increased FGF-CX gene expression, protein levels, orupregulated FGF-CX activity. In such clinical trials, the expression oractivity of FGF-CX and, preferably, other genes that have beenimplicated in, for example, a proliferative or neurological disorder,can be used as a “read out” or marker of the responsiveness of aparticular cell.

For example, genes, including FGF-CX, that are modulated in cells bytreatment with an agent (e.g., compound, drug or small molecule) thatmodulates FGF-CX activity (e.g., identified in a screening assay asdescribed herein) can be identified. Thus, to study the effect of agentson cellular proliferation disorders, for example, in a clinical trial,cells can be isolated and RNA prepared and analyzed for the levels ofexpression of FGF-CX and other genes implicated in the disorder. Thelevels of gene expression (i.e., a gene expression pattern) can bequantified by Northern blot analysis or RT-PCR, as described herein, oralternatively by measuring the amount of protein produced, by one of themethods as described herein, or by measuring the levels of activity ofFGF-CX or other genes. In this way, the gene expression pattern canserve as a marker, indicative of the physiological response of the cellsto the agent. Accordingly, this response state may be determined before,and at various points during, treatment of the individual with theagent.

In one embodiment, the invention provides a method for monitoring theeffectiveness of treatment of a subject with an agent (e.g., an agonist,antagonist, protein, peptide, nucleic acid, peptidomimetic, smallmolecule, or other drug candidate identified by the screening assaysdescribed herein) comprising the steps of (i) obtaining apre-administration sample from a subject prior to administration of theagent; (ii) detecting the level of expression of a FGF-CX protein, mRNA,or genomic DNA in the preadministration sample; (iii) obtaining one ormore post-administration samples from the subject; (iv) detecting thelevel of expression or activity of the FGF-CX protein, mRNA, or genomicDNA in the post-administration samples; (v) comparing the level ofexpression or activity of the FGF-CX protein, mRNA, or genomic DNA inthe pre-administration sample with the FGF-CX protein, mRNA, or genomicDNA in the post administration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly. For example,increased administration of the agent may be desirable to increase theexpression or activity of FGF-CX to higher levels than detected, i.e.,to increase the effectiveness of the agent. Alternatively, decreasedadministration of the agent may be desirable to decrease expression oractivity of FGF-CX to lower levels than detected, i.e., to decrease theeffectiveness of the agent.

Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant FGF-CX expression oractivity.

Diseases and disorders that are characterized by increased (relative toa subject not suffering from the disease or disorder) levels orbiological activity may be treated with Therapeutics that antagonize(i.e., reduce or inhibit) activity. Therapeutics that antagonizeactivity may be administered in a therapeutic or prophylactic manner.Therapeutics that may be utilized include, but are not limited to, (i) aFGF-CX polypeptide, or analogs, derivatives, fragments or homologsthereof; (ii) antibodies to a FGF-CX peptide; (iii) nucleic acidsencoding a FGF-CX peptide; (iv) administration of antisense nucleic acidand nucleic acids that are “dysfunctional” (i.e., due to a heterologousinsertion within the coding sequences of coding sequences to a FGF-CXpeptide) that are utilized to “knockout” endogenous function of a FGF-CXpeptide by homologous recombination (see, e.g., Capecchi, 1989, Science244: 1288-1292); or (v) modulators (i.e., inhibitors, agonists andantagonists, including additional peptide mimetic of the invention orantibodies specific to a peptide of the invention) that alter theinteraction between a FGF-CX peptide and its binding partner.

Diseases and disorders that are characterized by decreased (relative toa subject not suffering from the disease or disorder) levels orbiological activity may be treated with Therapeutics that increase (ie.,are agonists to) activity. Therapeutics that upregulate activity may beadministered in a therapeutic or prophylactic manner. Therapeutics thatmay be utilized include, but are not limited to, a FGF-CX peptide, oranalogs, derivatives, fragments or homologs thereof; or an agonist thatincreases bioavailability.

Increased or decreased levels can be readily detected by quantifyingpeptide and/or RNA, by obtaining a patient tissue sample (e.g., frombiopsy tissue) and assaying it in vitro for RNA or peptide levels,structure and/or activity of the expressed peptides (or mRNAs of aFGF-CX peptide). Methods that are well-known within the art include, butare not limited to, immunoassays (e.g., by Western blot analysis,immunoprecipitation followed by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/orhybridization assays to detect expression of mRNAs (e.g., Northernassays, dot blots, in situ hybridization, etc.).

In one aspect, the invention provides a method for preventing, in asubject, a disease or condition associated with an aberrant FGF-CXexpression or activity, by administering to the subject an agent thatmodulates FGF-CX expression or at least one FGF-CX activity. Subjects atrisk for a disease that is caused or contributed to by aberrant FGF-CXexpression or activity can be identified by, for example, any or acombination of diagnostic or prognostic assays as described herein.Administration of a prophylactic agent can occur prior to themanifestation of symptoms characteristic of the FGF-CX aberrancy, suchthat a disease or disorder is prevented or, alternatively, delayed inits progression. Depending on the type of FGF-CX aberrancy, for example,a FGF-CX agonist or FGF-CX antagonist agent can be used for treating thesubject. The appropriate agent can be determined based on screeningassays described herein.

Another aspect of the invention pertains to methods of modulating FGF-CXexpression or activity for therapeutic purposes. The modulatory methodof the invention involves contacting a cell with an agent that modulatesone or more of the activities of FGF-CX protein activity associated withthe cell. An agent that modulates FGF-CX protein activity can be anagent as described herein, such as a nucleic acid or a protein, anaturally-occurring cognate ligand of a FGF-CX protein, a peptide, aFGF-CX peptidomimetic, or other small molecule. In one embodiment, theagent stimulates one or more FGF-CX protein activity. Examples of suchstimulatory agents include active FGF-CX protein and a nucleic acidmolecule encoding FGF-CX that has been introduced into the cell. Inanother embodiment, the agent inhibits one or more FGF-CX proteinactivity. Examples of such inhibitory agents include antisense FGF-CXnucleic acid molecules and anti-FGF-CX antibodies. These modulatorymethods can be performed in vitro (e.g., by culturing the cell with theagent) or, alternatively, in vivo (e.g., by administering the agent to asubject). As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant expression or activity of a FGF-CX protein or nucleic acidmolecule. In one embodiment, the method involves administering an agent(e.g., an agent identified by a screening assay described herein), orcombination of agents that modulates (e.g., upregulates ordownregulates) FGF-CX expression or activity. In another embodiment, themethod involves administering a FGF-CX protein or nucleic acid moleculeas therapy to compensate for reduced or aberrant FGF-CX expression oractivity.

EXAMPLES Example 1 Molecular Cloning of the Sequence Encoding a FGF-CXProtein.

Oligonucleotide primers were designed for the amplification by PCR of aDNA segment, representing an open reading frame, coding for the fulllength FGF-CX. The forward primer includes a BglII restriction site(AGATCT) and a consensus Kozak sequence (CCACC). The reverse primercontains an in-frame XhoI restriction site for further subcloningpurposes. Both the forward and the reverse primers contain a 5′ clampsequence (CTCGTC). The sequences of the primers are the following:

FGF-CX-Forward: (SEQ ID NO:3) 5′ - CTCGTC AGATCT CCACC ATG GCT CCC TTAGCC GAA GTC - 3′ FGF-CX-Reverse: (SEQ ID NO:4) 5′ - CTCGTC CTCGAG AGTGTA CAT CAG TAG GTC CTT G - 3′

PCR reactions were set up using a total of 5 ng human prostate cDNAtemplate, 1 μM of each of the FGF-CX-Forward and FGF-CX-Reverse primers,5 micromoles dNTP (Clontech Laboratories, Palo Alto Calif.) and 1microliter of 50× Advantage-HF 2 polymerase (Clontech Laboratories, PaloAlto Calif.) in 50 microliter volume. The following PCR reactionconditions were used:

-   -   a) 96° C. 3 minutes    -   b) 96° C. 30 seconds denaturation    -   c) 70° C. 30 seconds, primer annealing. This temperature was        gradually decreased by 1° C./cycle.    -   d) 72° C. 1 minute extension.        Repeat steps (b)-(d) ten times    -   e) 96° C. 30 seconds denaturation    -   f) 60° C. 30 seconds annealing    -   g) 72° C. 1 minute extension        Repeat steps (e)-(g) 25 times    -   h) 72° C. 5 minutes final extension

A single PCR product, with the expected size of approximately 640 bp,was isolated from agarose gel and ligated into a pCR2.1 vector(Invitrogen, Carlsbad, Calif.). The cloned insert was sequenced usingvector specific M13 Forward(−40) and M13 Reverse primers, which verifiedthat the nucleotide sequence was 100% identical to the sequence in FIG.1 (SEQ ID NO:1) inserted directly between the upstream BglII cloningsite and the downstream XhoI cloning site. The cloned sequenceconstitutes an open reading frame coding for the predicted cgAB02085full length protein. The clone is called TA-cgAB02085-S274-F19.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. In thecase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

Equivalents

From the foregoing detailed description of the specific embodiments ofthe invention, it should be apparent that particular novel compositionsand methods involving nucleic acids, polypeptides, antibodies, detectionand treatment have been described. Although these particular embodimentshave been disclosed herein in detail, this has been done by way ofexample for purposes of illustration only, and is not intended to belimiting with respect to the scope of the appended claims that follow.In particular, it is contemplated by the inventors that varioussubstitutions, alterations, and modifications may be made as a matter ofroutine for a person of ordinary skill in the art to the inventionwithout departing from the spirit and scope of the invention as definedby the claims. Indeed, various modifications of the invention inaddition to those described herein will become apparent to those skilledin the art from the foregoing description and accompanying figures. Suchmodifications are intended to fall within the scope of the appendedclaims.

1. An isolated nucleic acid molecule comprising a nucleotide sequenceencoding a polypeptide comprising the amino acid sequence of SEQ IDNO:2, or the complement of said nucleic acid molecule, wherein theencoded polypeptide has cell proliferation stimulatory activity.
 2. Thenucleic acid molecule of claim 1, wherein the nucleic acid moleculecomprises the nucleotide sequence of SEQ ID NO:1.
 3. The nucleic acidmolecule of claim 1, wherein the nucleotide sequence encodes apolypeptide consisting of the amino acid sequence of SEQ ID NO:2,wherein the polypeptide has cell proliferation stimulatory activity. 4.A nucleic acid vector comprising the nucleic acid molecule of claim 1.5. The nucleic acid vector of claim 4, wherein said vector is anexpression vector.
 6. The vector of claim 4, further comprising aregulatory element operably linked to said nucleic acid molecule.
 7. Anisolated host cell comprising the isolated nucleic acid molecule ofclaim
 1. 8. A method of producing an isolated polypeptide of SEQ IDNO:2, said method comprising the step of culturing the host cell ofclaim 7, under conditions suitable for expression of the coding strandof the nucleic acid molecule encoding said polypeptide of SEQ ID NO:2and recovering the polypeptide.
 9. A composition comprising the nucleicacid of claim 1, and a pharmaceutically acceptable carrier.
 10. A kitcomprising in one or more containers, the composition of claim
 9. 11. Anisolated nucleic acid molecule comprising a nucleotide sequence encodinga variant of the amino acid sequence of SEQ ID NO:2, wherein saidvariant comprises one or more conservative amino acid substitutions ascompared to the amino acid sequence of SEQ ID NO:2, wherein saidsubstitutions do not alter the cell proliferation stimulatory activityof the encoded polypetide, and wherein the nucleic acid molecule encodesa variant polypeptide at least 95% identical to the polypeptidecomprising the amino acid sequence of SEQ ID NO:2.